Lipid nanoparticle formulations

ABSTRACT

Improved formulations of lipid nanoparticles are provided. Use of the lipid nanoparticles for delivery of a therapeutic agent and methods for their preparation are also provided.

BACKGROUND Technical Field

Embodiments of the present invention generally relate to optimizedformulations of lipid nanoparticles useful for facilitating theintracellular delivery of therapeutic agents, such as nucleic acids(e.g., oligonucleotides, messenger RNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipid formulations and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticle formulations would provide optimal drug:lipid ratios,protect the nucleic acid from degradation and clearance in serum, besuitable for systemic or local delivery, and provide intracellulardelivery of the nucleic acid. In addition, these lipid-nucleic acidparticles should be well-tolerated and provide an adequate therapeuticindex, such that patient treatment at an effective dose of the nucleicacid is not associated with unacceptable toxicity and/or risk to thepatient. The present invention provides these and related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention provide optimized lipidnanoparticle (LNP) formulation. Exemplary LNPs comprise cationiclipid(s), a neutral lipid, a steroid, a polymer conjugated lipid, and atherapeutic agent, or a pharmaceutically acceptable salt thereof,encapsulated within or associated with the LNP. LNPs known in the arttypically comprise higher concentrations of cationic lipids (e.g., >50mol percent), a single cationic lipid or cationic lipids with adifferent pKa than embodiments of the present disclosure. In contrast tosuch prior art LNPs, the present Applicant has unexpectedly discoveredthat characteristics of the optimized formulations of the presentdisclosure provide important improvements for LNP properties related todelivery of the therapeutic agent (e.g., increased stability andenhanced delivery).

In some instances, the lipid nanoparticles are used to deliver nucleicacids such as antisense and/or messenger RNA.

Accordingly, in one embodiment is provided a lipid nanoparticlecomprising:

-   -   i) between 40 and 50 mol percent of a cationic lipid;    -   ii) a neutral lipid;    -   iii) a steroid;    -   iv) a polymer conjugated lipid; and    -   v) a therapeutic agent, or a pharmaceutically acceptable salt        thereof, encapsulated within or associated with the lipid        nanoparticle.

Another embodiment provides a lipid nanoparticle comprising:

-   -   i) a cationic lipid having an effective pKa greater than 6.0;    -   ii) from 5 to 15 mol percent of a neutral lipid;    -   iii) from 1 to 15 mol percent of an anionic lipid;    -   iv) from 30 to 45 mol percent of a steroid;    -   v) a polymer conjugated lipid; and    -   vi) a therapeutic agent, or a pharmaceutically acceptable salt        thereof, encapsulated within or associated with the lipid        nanoparticle,

wherein the mol percent is determined based on total mol of lipidpresent in the lipid nanoparticle.

One other embodiment provides a lipid nanoparticle comprising:

-   -   i) a first cationic lipid having a first effective pKa;    -   ii) a second cationic lipid having a second effective pKa, the        second effective pKa being greater than the first effective pKa;    -   iii) a neutral lipid;    -   iv) a steroid;    -   v) a polymer conjugated lipid; and    -   vi) a therapeutic agent, or a pharmaceutically acceptable salt        or prodrug thereof, encapsulated within or associated with the        lipid nanoparticle,

wherein the lipid nanoparticle has an effective pKa between the firstand second effective pKa's.

Pharmaceutical compositions comprising the disclosed lipid nanoparticlesand methods for use of the same for treatment of various diseases orconditions, such as those caused by infectious entities and/orinsufficiency of a protein, are also provided.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising administering the disclosed lipid nanoparticles, orpharmaceutical composition comprising the same, to the patient.

These and other aspects of embodiments of the invention will be apparentupon reference to the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIGS. 1A and 1B provide luciferase activity data as a function of N/PRatio for representative lipids in liver and spleen tissue,respectively.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Embodiments of the present invention are based, in part, upon thediscovery that modulating aspects of the cationic lipid content and/oreffective pKa of a lipid nanoparticle provides surprisingly superiorcharacteristics. Namely, certain unpredicted modifications (e.g.,concentration of cationic lipid, including an additional distinctcationic lipid and/or including an anionic lipid) surprisingly result inimproved properties of a lipid nanoparticle as a delivery vehicle fortherapeutic agents (e.g., nucleic acids).

Accordingly, in some embodiments, lipid nanoparticles (LNPs) comprising,inter alia, between 40 and 50 mol percent of a cationic lipid provideunexpected advantages over previously known LNPs when used for deliveryof an active or therapeutic agent, such as a nucleic acid, into a cellof a mammal. In particular, one embodiment provides a lipid nanoparticlecomprising between 40 and 50 mol percent of a cationic lipid, a neutrallipid, a steroid, a polymer conjugated lipid, and a therapeutic agentencapsulated within or associated with the lipid nanoparticle.

In some embodiments, the LNPs comprise, inter alia, first and secondcationic lipids, each of the first and second cationic lipids having adifferent effective pKa. Other known LNPs comprise only one type ofcationic lipid and the effective pKa of the LNP is thus highly dependenton use of cationic lipids having an optimized effective pKa. However,the data provided herein demonstrate the surprising result that cationiclipids which would have previously been excluded from use in LNPs due toan undesirable effective pKa can be admixed with other cationic lipidshaving a different effective pKa to form an LNP having an optimizedeffective pKa. Specifically, the present inventors have discovered thatLNPs comprising, inter alia, first and second cationic lipids, each ofthe first and second cationic lipids having a different effective pKa,have surprisingly better properties for use as a delivery vehicle fortherapeutic agents, such as nucleic acids, relative to LNPs comprisingonly the first or the second cationic lipid.

In addition, the present inventors have discovered that LNPs comprising,inter alia, a cationic lipid having an effective pKa greater than 6.0and from 1 to 15 mol percent of an anionic lipid, have surprisinglyimproved properties for use as a delivery vehicle. Embodiments of theLNPs described herein provide increased encapsulation efficiency and/orincreased in vivo activity, resulting in a significant increase in thetherapeutic index as compared to LNPs previously described.

In particular embodiments, the present invention provides lipidnanoparticles for the in vitro and in vivo delivery of mRNA and/or otheroligonucleotides. In some embodiments, these improved lipid nanoparticlecompositions are useful for expression of protein encoded by mRNA. Inother embodiments, these improved lipid nanoparticles are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticles are useful for inducing apharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles of embodiments of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, embodiments of thepresent invention provide a method for administering a therapeutic agentto a patient in need thereof, the method comprising administering alipid nanoparticle as described herein to the patient.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles of embodiments of the present invention may be usedto induce expression of a desired protein both in vitro and in vivo bycontacting cells with a lipid nanoparticle. The expressed protein mayhave a biological effect, such as inducing an immune response.Alternatively, the lipid nanoparticles and compositions of embodimentsof the present invention may be used to decrease the expression oftarget genes and proteins both in vitro and in vivo by contacting cellswith a lipid nanoparticle. The lipid nanoparticles and compositions ofembodiments of the present invention may also be used for co-delivery ofdifferent nucleic acids (e.g., mRNA and plasmid DNA) separately or incombination, such as may be useful to provide an effect requiringcolocalization of different nucleic acids (e.g. mRNA encoding for asuitable gene modifying enzyme and DNA segment(s) for incorporation intothe host genome).

Nucleic acids for use with embodiments of this invention may be preparedaccording to any available technique. For mRNA, the primary methodologyof preparation is, but not limited to, enzymatic synthesis (also termedin vitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g. including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L. and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012).

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (e.g., ethanol,isopropanol) in the presence of monovalent cations or lithium chloride.Additional, non-limiting examples of purification procedures which canbe used include size exclusion chromatography (Lukaysky, P. J. andPuglisi, J. D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H.,Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA fortherapy: HPLC purification eliminates immune activation and improvestranslation of nucleoside-modified, protein-encoding mRNA, Nucl AcidRes, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K.,HPLC Purification of in vitro transcribed long RNA in SyntheticMessenger RNA and Cell Metabolism Modulation in Methods in MolecularBiology v. 969 (Rabinovich, P. H. Ed), 2013). HPLC purified mRNA hasbeen reported to be translated at much greater levels, particularly inprimary cells and in vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e., cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see, e.g., Grudzien-Nogalska, E.,Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E.,Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs withsuperior translation and stability properties in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see e.g. Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K.,In vitro transcription of long RNA containing modified nucleosides inSynthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840. The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see, e.g., U.S. Pub. No. 2012/0251618). In vitro synthesis ofnucleoside-modified mRNA have been reported to have reduced ability toactivate immune sensors with a concomitant enhanced translationalcapacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see, e.g., Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisinvention. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

For plasmid DNA, preparation for use with this invention commonlyutilizes but is not limited to expansion and isolation of the plasmidDNA in vitro in a liquid culture of bacteria containing the plasmid ofinterest. The presence of a gene in the plasmid of interest that encodesresistance to a particular antibiotic (penicillin, kanamycin, etc.)allows those bacteria containing the plasmid of interest to selectivelygrow in antibiotic-containing cultures. Methods of isolating plasmid DNAare widely used and well known in the art (see, e.g., Heilig, J.,Elbing, K. L. and Brent, R (2001) Large-Scale Preparation of PlasmidDNA. Current Protocols in Molecular Biology. 41:11:1.7:1.7.1-1.7.16;Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, S.R. (2008), Large-scale production of endotoxin-free plasmids fortransient expression in mammalian cell culture. Biotechnol. Bioeng., 99:557-566; and U.S. Pat. No. 6,197,553 B1). Plasmid isolation can beperformed using a variety of commercially available kits including, butnot limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo)and PureYield MaxiPrep (Promega) kits as well as with commerciallyavailable reagents.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g., asample of cells in culture expressing the desired protein) or a testmammal (e.g., a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g., nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g. a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model) that is not contacted with oradministered the nucleic acid. When the desired protein is present in acontrol sample or a control mammal, the expression of a desired proteinin a control sample or a control mammal may be assigned a value of 1.0.In particular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g., a sample of cells in culture expressing the target gene) or atest mammal (e.g., a mammal such as a human or an animal model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animalmodel such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g. an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

An “anionic lipid” refers to a lipid capable of being negativelycharged. Exemplary anionic lipids include one or more phosphate group(s)which bear a negative charge, for example at physiological pHs. In someembodiments, the anionic lipid does not include a serine moiety,including phosphatidylserine lipids.

“Phosphatidylglycerol lipid” refers to a lipid with a structure thatgenerally comprises a glycerol 3-phosphate backbone which is attached tosaturated or unsaturated fatty acids via and ester linkage. Exemplaryphosphatidylglycerol lipids have the following structure:

wherein R₁ and R₂ are each independently a branched or straight,saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl).

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range, e.g., pH ˜3to pH ˜9. Charged lipids may be synthetic or naturally derived. Examplesof charged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates,dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g., DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of Formula I, II, III, IV or V or otherspecified cationic lipids. In some embodiments, lipid nanoparticles areincluded in a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles of the invention comprise a nucleicacid. Such lipid nanoparticles typically comprise a compound of FormulaIII, III, IV or V and one or more excipient selected from neutrallipids, charged lipids, steroids and polymer conjugated lipids. In someembodiments, the active agent or therapeutic agent, such as a nucleicacid, may be encapsulated in the lipid portion of the lipid nanoparticleor an aqueous space enveloped by some or all of the lipid portion of thelipid nanoparticle, thereby protecting it from enzymatic degradation orother undesirable effects induced by the mechanisms of the host organismor cells, e.g., an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipids and lipid nanoparticlescomprising nucleic acids and their method of preparation are disclosedin, e.g., U.S. Pat. Nos. 8,569,256, 5,965,542 and U.S. PatentPublication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708,2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373,2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338,2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411,2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527,2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032,2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910,2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025,2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2017/004143,WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO2013/086322, WO 2013/016058, WO 2013/086373, WO2011/141705, and WO2001/07548, the full disclosures of which are herein incorporated byreference in their entirety for all purposes.

Other exemplary lipids and lipid nanoparticles and their manufacture aredescribed in the art, for example in U.S. Patent Application PublicationNo. U.S. 2012/0276209, Semple et al., 2010, Nat Biotechnol.,28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha etal., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys ChemC Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int JCancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleicAcids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier etal., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013,Nanomedicine, 9(5): 665-74, each of which are incorporated by referencein their entirety. Lipids and their manufacture can be found, forexample, in U.S. Pub. No. 2015/0376115 and 2016/0376224, both of whichare incorporated herein by reference.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Amino acid” refers to naturally-occurring and non-naturally occurringamino acids. An amino acid lipid can be made from a genetically encodedamino acid, a naturally occurring non-genetically encoded amino acid, ora synthetic amino acid. Examples of amino acids include Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, and Val. Examples of amino acids also include azetidine,2-aminooctadecanoic acid, 2-aminoadipic acid, 3-aminoadipic acid,2,3-diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyricacid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2′-diaminopimelicacid, 6-aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid,desmosine, omithine, citrulline, N-methylisoleucine, norleucine,tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sacrosine,N-ethylglycine, cyclohexylglycine, 4-oxo-cyclohexylglycine,N-ethylasparagine, cyclohexylalanine, t-butylalanine, naphthylalanine,pyridylalanine, 3-chloroalanine, 3-benzothienylalanine,4-halophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,2-thienylalanine, methionine, methionine sulfoxide, homoarginine,norarginine, nor-norarginine, N-acetyllysine, 4-aminophenylalanine,N-methylvaline, homocysteine, homoserine, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, 6—N-methyllysine, norvaline, 0-allyl-serine,0-allyl-threonine, alpha-aminohexanoic acid, alpha-aminovaleric acid,pyroglutamic acid, and derivatives thereof. “Amino acid” includes alpha-and beta-amino acids. Examples of amino acid residues can be found inFasman, CRC Practical Handbook of Biochemistry and Molecular Biology,CRC Press, Inc. (1989).

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double (alkenyl) and/or triplebonds (alkynyl)), having, for example, from one to twenty-four carbonatoms (C₁-C₂₄ alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six tosixteen carbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉alkyl), one to fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbonatoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one tosix carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl(iso propyl), n butyl, n pentyl, 1,1-dimethylethyl (t butyl),3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl,pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl,hexynyl, and the like. Unless stated otherwise specifically in thespecification, an alkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double (alkenylene) and/ortriple bonds (alkynylene)), and having, for example, from one totwenty-four carbon atoms (C₁-C₂₄ alkylene), one to fifteen carbon atoms(C₁-C₁₅ alkylene),one to twelve carbon atoms (C₁-C₁₂ alkylene), one toeight carbon atoms (C₁-C₈ alkylene), one to six carbon atoms (C₁-C₆alkylene), two to four carbon atoms (C₂-C₄ alkylene), one to two carbonatoms (C₁-C₂ alkylene), e.g., methylene, ethylene, propylene,n-butylene, ethenylene, propenylene, n-butenylene, propynylene,n-butynylene, and the like. The alkylene chain is attached to the restof the molecule through a single or double bond and to the radical groupthrough a single or double bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, an alkylene chain may beoptionally substituted.

The term “alkenyl” refers to an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike.

“Alkoxy” refers to an alkyl, cycloalkyl, alkenyl, or alkynyl groupcovalently bonded to an oxygen atom.

“Alkanoyloxy” refers to —O—C(═O)-alkyl groups.

“Alkylamino” refers to the group —NRR′, where R and R′ are each eitherhydrogen or alkyl, and at least one of R and R′ is alkyl. Alkylaminoincludes groups such as piperidino wherein R and R′ form a ring. Theterm “alkylaminoalkyl” refers to -alkyl-NRR′.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The terms “acyl,” “carbonyl,” and “alkanoyl” refer to any alkyl,alkenyl, or alkynyl wherein the carbon at the point of attachment issubstituted with an oxo group, as defined below. The following arenon-limiting examples of acyl, carbonyl or alkanoyl groups: —C(═O)alkyl,—C(═O)alkenyl, and —C(═O)alkynyl.

“Aryl” refers to any stable monocyclic, bicyclic, or polycyclic carbonring system of from 4 to 12 atoms in each ring, wherein at least onering is aromatic. Some examples of an aryl include phenyl, naphthyl,tetrahydro-naphthyl, indanyl, and biphenyl. Where an aryl substituent isbicyclic and one ring is non-aromatic, it is understood that attachmentis to the aromatic ring. An aryl may be substituted or unsubstituted.

“Carboxyl” refers to a functional group of the formula —C(═O)OH.

“Cyano” refers to a functional group of the formula —CN.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise statedspecifically in the specification, a cycloalkylene group may beoptionally substituted.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, both of which have independently between 2 and 30 carbonsbonded to the 1- and 2-position of glycerol by ester linkages. The acylgroups can be saturated or have varying degrees of unsaturation.Suitable acyl groups include, but are not limited to, lauroyl (C12),myristoyl (C14), palmitoyl (C16), stearoyl (C18), and icosoyl (C20). Inpreferred embodiments, the fatty acid acyl chains of one compound arethe same, i.e., both myristoyl (i.e., dimyristoyl), both stearoyl (i.e.,distearoyl), etc.

The term “heterocycle” or “heterocyclyl” refers to an aromatic ornonaromatic ring system of from five to twenty-two atoms, wherein from 1to 4 of the ring atoms are heteroatoms selected from oxygen, nitrogen,and sulfur. Thus, a heterocycle may be a heteroaryl or a dihydro ortetrathydro version thereof. Heterocycles include, but are not limitedto, pyrrolidine, tetryhydrofuran, thiolane, azetidine, oxetane,thietane, diazetidine, dioxetane, dithietane, piperidine,tetrahydrofuran, pyran, tetrahydropyran, thiacyclohexane,tetrahydrothiophene, pyridine, pyrimidine and the like.

“Heteroaryl” refers to any stable monocyclic, bicyclic, or polycycliccarbon ring system of from 4 to 12 atoms in each ring, wherein at leastone ring is aromatic and contains from 1 to 4 heteroatoms selected fromoxygen, nitrogen and sulfur. Some examples of a heteroaryl includeacridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,pyrrolyl, and tetrahydroquinolinyl. A heteroaryl includes the N-oxidederivative of a nitrogen-containing heteroaryl.

The terms “alkylamine” and “dialkylamine” refer to —NH(alkyl) and—N(alkyl)₂ radicals respectively.

The term “alkylphosphate” refers to —O—P(Q′)(Q″)—O—R, wherein Q′ and Q″are each independently O, S, N(R)₂, optionally substituted alkyl oralkoxy; and R is optionally substituted alkyl, ω-aminoalkyl orω-(substituted)aminoalkyl.

The term “alkylphosphorothioate” refers to an alkylphosphate wherein atleast one of Q′ or Q″ is S.

The term “alkylphosphonate” refers to an alkylphosphate wherein at leastone of Q′ or Q″ is alkyl.

“Hydroxyalkyl” refers to an —O-alkyl radical.

The term “alkylheterocycle” refers to an alkyl where at least onemethylene has been replaced by a heterocycle.

The term “ω-aminoalkyl” refers to -alkyl-NH₂ radical. And the term“ω-(substituted)aminoalkyl refers to an ω-aminoalkyl wherein at leastone of the H on N has been replaced with alkyl.

The term “ω-phosphoalkyl” refers to -alkyl-O—P(Q′)(Q″)—O—R, wherein Q′and Q″ are each independently O or S and R optionally substituted alkyl.

The term “ω-thiophosphoalkyl” refers to ω-phosphoalkyl wherein at leastone of Q′ or Q″ is S.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, cycloalkyl or cycloalkylene) wherein at least onehydrogen atom is replaced by a bond to a non-hydrogen atom such as, butnot limited to: a halogen atom such as F, Cl, Br, or I; oxo groups (═O);hydroxyl groups (—OH); C₁-C₁₂ alkyl groups; cycloalkyl groups;—(C═O)OR′; —O(C═O)R′; —C(═O)R′; —OR′; —S(O)_(x)R′; —C(═O)SR′; —SC(═O)R′;—NR′R′; —NR′C(═O)R′; —C(═O)NR′R′; —NR′C(═O)NR′R′; —OC(═O)NR′R′;—NR′C(═O)OR′; —NR′S(O)_(x)NR′R′; —NR′S(O)_(x)R′; and —S(O)_(x)NR′R′,wherein: R′ is, at each occurrence, independently H, C₁-C₁₅ alkyl orcycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is aC₁-C₁₂ alkyl group. In other embodiments, the substituent is acycloalkyl group. In other embodiments, the substituent is a halo group,such as fluoro. In other embodiments, the substituent is an oxo group.In other embodiments, the substituent is a hydroxyl group. In otherembodiments, the substituent is an alkoxy group (—OR′). In otherembodiments, the substituent is a carboxyl group. In other embodiments,the substituent is an amine group (—NR′R′).

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound, such as a therapeutic agent,that may be converted under physiological conditions or by solvolysis toa biologically active compound of the invention. Thus, the term“prodrug” refers to a metabolic precursor of a compound of the inventionthat is pharmaceutically acceptable. A prodrug may be inactive whenadministered to a subject in need thereof, but is converted in vivo toan active compound of the invention. Prodrugs are typically rapidlytransformed in vivo to yield the parent compound of the invention, forexample, by hydrolysis in blood. The prodrug compound often offersadvantages of solubility, tissue compatibility or delayed release in amammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp.7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is providedin Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, Ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs(e.g., a prodrug of a therapeutic agent) may be prepared by modifyingfunctional groups present in the compound of the invention in such a waythat the modifications are cleaved, either in routine manipulation or invivo, to the parent compound of the invention. Prodrugs includecompounds wherein a hydroxy, amino or mercapto group is bonded to anygroup such that, when the prodrug is administered to a mammaliansubject, cleaves to form a free hydroxy, free amino or free mercaptogroup, respectively. Examples of prodrugs include, but are not limitedto, acetate, formate and benzoate derivatives of alcohol or amidederivatives of amine functional groups in the therapeutic agents of theinvention and the like.

Embodiments of the invention disclosed herein are also meant toencompass all pharmaceutically acceptable lipid nanoparticles andcomponents thereof (e.g., cationic lipid, therapeutic agent, etc.) beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabeledLNPs could be useful to help determine or measure the effectiveness ofthe compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled LNPs, for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, i.e., ³H,and carbon-14, that is, ¹⁴C, are particularly useful for this purpose inview of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, that is, ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof Formula I, II, III, IV or V can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Examples as set out below using an appropriateisotopically-labeled reagent in place of the non-labeled reagentpreviously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

A “pharmaceutical composition” refers to a formulation of an LNP of theinvention and a medium generally accepted in the art for the delivery ofthe biologically active compound to mammals, e.g., humans. Such a mediumincludes all pharmaceutically acceptable carriers, diluents orexcipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

Lipid Nanoparticles

In certain embodiments, the present invention provides lipidnanoparticles comprising a therapeutic agent encapsulated within orassociated with the lipid nanoparticle. The conventional knowledge inthe art is that the most effective LNP formulation is one where thecationic lipid concentration is maximized with respect to the other LNPcomponents. For example, prior art LNPs typically comprise a cationiclipid at molar ratios of 50% or more since maximal cationic lipidconcentration was thought to be essential to obtaining desirableencapsulation efficiency, in vivo activity and other characteristics ofLNPs. However, the data provided herein demonstrate the surprisingresult that the most effective cationic lipid proportion is notnecessarily the maximum concentration accommodated and the performanceof LNPs can be improved with other unpredicted combinations with chargedlipid species (e.g., cationic lipids with lower pKa, an additionalcationic lipid or an anionic lipid).

Accordingly, in one embodiment (“Embodiment 1”) is provided a lipidnanoparticle comprising:

i) between 40 and 50 mol percent of a cationic lipid;

ii) a neutral lipid;

iii) a steroid;

iv) a polymer conjugated lipid; and

v) a therapeutic agent encapsulated within or associated with the lipidnanoparticle.

As used herein, “mol percent” refers to a component's molar percentagerelative to total mols of all lipid components in the LNP (i.e., totalmols of cationic lipid(s), the neutral lipid, the steroid and thepolymer conjugated lipid).

In certain aspects of Embodiment 1, the lipid nanoparticle comprisesfrom 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 molpercent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45to 48 mol percent, from 46 to 48 mol percent, or from 47.2 to 47.8 molpercent of the cationic lipid. In certain specific embodiments, thelipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5,47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.

In certain other embodiments of Embodiment 1, the neutral lipid ispresent in a concentration ranging from 5 to 15 mol percent, 7 to 13 molpercent, or 9 to 11 mol percent. In certain specific embodiments, theneutral lipid is present in a concentration of about 9.5, 10 or 10.5 molpercent. In some embodiments, the molar ratio of the cationic lipid tothe neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, from about4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to 4.8:1.0.

In different embodiments of Embodiment 1, the steroid is cholesterol. Insome embodiments, the steroid is present in a concentration ranging from39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molarpercent, from 40 to 42 molar percent, from 42 to 44 molar percent, orfrom 44 to 46 molar percent. In certain specific embodiments, thesteroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46molar percent. In certain embodiments, the molar ratio of cationic lipidto the steroid ranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to1.0:1.2. In different embodiments, the steroid is cholesterol.

In other embodiments of Embodiment 1, the mRNA to lipid ratio in the LNP(i.e., N/P, were N represents the moles of cationic lipid and Prepresents the moles of phosphate present as part of the nucleic acidbackbone) range from 2:1 to 30:1, for example 3:1 to 22:1. In otherembodiments, N/P ranges from 6:1 to 20:1 or 2:1 to 12:1. Exemplary N/Pranges include about 3:1. About 6:1, about 12:1 and about 22:1.

Another embodiment (“Embodiment 2”) provides a lipid nanoparticlecomprising:

i) a cationic lipid having an effective pKa greater than 6.0;

ii) from 5 to 15 mol percent of a neutral lipid;

iii) from 1 to 15 mol percent of an anionic lipid;

iv) from 30 to 45 mol percent of a steroid;

v) a polymer conjugated lipid; and

vi) a therapeutic agent, or a pharmaceutically acceptable salt orprodrug thereof, encapsulated within or associated with the lipidnanoparticle,

wherein the mol percent is determined based on total mol of lipidpresent in the lipid nanoparticle.

In certain embodiments of Embodiment 2, the cationic lipid can be any ofa number of lipid species which carry a net positive charge at aselected pH, such as physiological pH. Exemplary cationic lipids aredescribed herein below. In some embodiments, the cationic lipid has apKa greater than 6.25. In other embodiments, the cationic lipid has apKa greater than 6.5. In certain embodiments, the cationic lipid has apKa greater than 6.1, greater than 6.2, greater than 6.3, greater than6.35, greater than 6.4, greater than 6.45, greater than 6.55, greaterthan 6.6, greater than 6.65, or greater than 6.7.

In other embodiments of Embodiment 2, the lipid nanoparticle comprisesfrom 40 to 45 mol percent of the cationic lipid. In some embodiments,the lipid nanoparticle comprises from 45 to 50 mole percent of thecationic lipid.

In various embodiments, the molar ratio of the cationic lipid to theneutral lipid ranges from about 2:1 to about 8:1. In some embodiments,the lipid nanoparticle comprises from 5 to 10 mol percent of the neutrallipid.

Exemplary anionic lipids for use in Embodiment 2 include, but are notlimited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG) or1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG).

In some embodiments of Embodiment 2, the lipid nanoparticle comprisesfrom 1 to 10 mole percent of the anionic lipid. In certain embodiments,the lipid nanoparticle comprises from 1 to 5 mole percent of the anioniclipid. In some embodiments, the lipid nanoparticle comprises from 1 to 9mole percent, from 1 to 8 mole percent, from 1 to 7 mole percent, orfrom 1 to 6 mole percent of the anionic lipid. In certain embodiments,the mol ratio of anionic lipid to neutral lipid ranges from 1:1 to 1:10.

In certain embodiments of Embodiment 2, the steroid cholesterol. In someof these embodiments, the molar ratio of the cationic lipid tocholesterol ranges from about 5:1 to 1:1. In certain embodiments, thelipid nanoparticle comprises from 32 to 40 mol percent of the steroid.

In certain other embodiments of Embodiment 2, the sum of the mol percentof neutral lipid and mol percent of anionic lipid ranges from 5 to 15mol percent. In certain embodiments, wherein the sum of the mol percentof neutral lipid and mol percent of anionic lipid ranges from 7 to 12mol percent.

In different embodiments of Embodiment 2, the mol ratio of anionic lipidto neutral lipid ranges from 1:1 to 1:10. In some embodiments, the sumof the mol percent of neutral lipid and mol percent steroid ranges from35 to 45 mol percent.

In some more specific examples of Embodiment 2, the lipid nanoparticlecomprises:

-   -   i) from 45 to 55 mol percent of the cationic lipid;    -   ii) from 5 to 10 mol percent of the neutral lipid;    -   iii) from 1 to 5 mol percent of the anionic lipid; and    -   iv) from 32 to 40 mol percent of the steroid.

In certain other embodiments of Embodiment 2, the lipid nanoparticlecomprises from 1.0 to 2.5 mol percent of the conjugated lipid. In someembodiments, the polymer conjugated lipid is present in a concentrationof about 1.5 mol percent.

Still another embodiment (“Embodiment 3”) provides a lipid nanoparticlecomprising:

i) a first cationic lipid having a effective pKa;

ii) a second cationic lipid having a second effective pKa, the secondeffective pKa being greater than the first effective pKa;

iii) a neutral lipid;

iv) a steroid;

v) a polymer conjugated lipid; and

vi) a therapeutic agent, or a pharmaceutically acceptable salt thereof,encapsulated within or associated with the lipid nanoparticle,

wherein the lipid nanoparticle has an effective pKa between the firstand second effective pKa's.

In embodiments of Embodiment 3, the first effective pKa is less than5.50, less than 5.60, less than 5.70, less than 5.80, less than 5.90 orless than 6.0. In other embodiments, the first effective pKa is lessthan 5.55, less than 5.65, less than 5.75, less than 5.85 or less than5.95. In some embodiments, the first effective pKa ranges from about 5.5to about 6.3.

In other embodiments of Embodiment 3, the second effective pKa rangesfrom about 6.3 to about 7. In different embodiments, the secondeffective pKa is greater than 6.25, greater than 6.35, greater than6.45, greater than 6.55, greater than 6.65 or greater than 6.75. Inother embodiments, the second effective pKa is greater than 6.30,greater than 6.40, greater than 6.50, greater than 6.60 or greater than6.70.

The lipid nanoparticle of various embodiments of Embodiment 3 has anoptimized effective pKa. For example, in some embodiments the lipidnanoparticle has an effective pKa ranging from 5.5 to 6.5, from 5.9 to6.4, from 5.9 to 6.35 or from 6.0 to 6.2.

In certain embodiments of Embodiment 3, the total mol percent ofcationic lipid in the lipid nanoparticle ranges from 40 to 55 molpercent or from 45 to 55 mol percent based on total lipid present in thelipid nanoparticle. As used herein, “mol percent” refers to acomponent's molar percentage relative to total mols of all lipidcomponents in the LNP (i.e., total mols of cationic lipid, the neutrallipid, the steroid and the polymer conjugated lipid).

In other embodiments of Embodiment 3, the mol ratio of the firstcationic lipid to the second cationic lipid ranges from 1:9 to 1:2 orfrom 1:20 to 1:2.

The first and second cationic lipids of Embodiment 3 can independentlybe any of a number of lipid species which carry a net positive charge ata selected pH, such as physiological pH. Such lipids include, but arenot limited to the lipids disclosed herein below.

In certain embodiments of Embodiment 3, the neutral lipid is present ina concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent,or 9 to 11 mol percent. In certain specific embodiments, the neutrallipid is present in a concentration of about 9.5, 10 or 10.5 molpercent. In some embodiments, the molar ratio of the cationic lipid tothe neutral lipid ranges from about 4.1:1.0 to about 4.9:1.0, from about4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to 4.8:1.0.

In certain other embodiments of Embodiment 3, the steroid ischolesterol. In some embodiments, the steroid is present in aconcentration ranging from 39 to 49 molar percent, 40 to 46 molarpercent, from 40 to 44 molar percent, from 40 to 42 molar percent, from42 to 44 molar percent, or from 44 to 46 molar percent. In certainspecific embodiments, the steroid is present in a concentration of 40,41, 42, 43, 44, 45, or 46 molar percent. In certain embodiments, themolar ratio of cationic lipid to the steroid ranges from 1.0:0.9 to1.0:1.2, or from 1.0:1.0 to 1.0:1.2.

In some embodiments of Embodiment 3, the molar ratio of total cationiclipid (i.e., the sum of the first and second cationic lipid) to steroidranges from 5:1 to 1:1.

In any of Embodiment 1, 2 or 3, the lipid nanoparticle comprises from1.0 to 2.5 mol percent of the conjugated lipid. In some embodiments, thepolymer conjugated lipid is present in a concentration of about 1.5 molpercent.

In various embodiments of Embodiments 1, 2 or 3, the molar ratio ofcationic lipid to polymer conjugated lipid ranges from about 100:1 toabout 20:1. In some embodiments, the molar ratio of cationic lipid tothe polymer conjugated lipid ranges from about 35:1 to about 25:1.

In other of any of Embodiments 1, 2 or 3, the molar ratio of totalcationic lipid to polymer conjugated lipid ranges from about 100:1 toabout 20:1. In some embodiments, the molar ratio of total cationic lipidto the polymer conjugated lipid ranges from about 35:1 to about 25:1.

In some of Embodiments 1, 2 or 3, the lipid nanoparticle has a meandiameter ranging from 50 nm to 100 nm, or from 60 nm to 85 nm.

The cationic lipid(s) for use in any of Embodiments 1, 2 or 3 can be anyof a number of lipid species which carry a net positive charge at aselected pH, such as physiological pH. Such lipids include, but are notlimited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N-(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE).

Additionally, a number of commercial preparations of cationic lipids areavailable which can be used in Embodiments 1, 2 or 3. These include, forexample, LIPOFECTIN® (commercially available cationic liposomescomprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), fromGIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially availablecationic liposomes comprisingN-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

In one specific embodiment, the cationic lipid for use in Embodiments 1,2 or 3 is independently an amino lipid. Suitable amino lipids includethose described in WO 2012/016184, incorporated herein by reference inits entirety. Representative amino lipids include, but are not limitedto, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

In some of Embodiments 1, 2 or 3, a cationic lipid (e.g., the cationiclipid, the first cationic lipid, the second cationic lipid) has thefollowing formula:

wherein R₁ and R₂ are either the same or different and independentlyoptionally substituted C₁₀-C₂₄ alkyl, optionally substituted C₁₀-C₂₄alkenyl, optionally substituted C₁₀-C₂₄ alkynyl, or optionallysubstituted C₁₀-C₂₄ acyl;

R₃ and R₄ are either the same or different and independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, oroptionally substituted C₂-C₆ alkynyl or R₃ and R₄ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen;

R₅ is either absent or present and when present is hydrogen or C₁-C₆alkyl; m, n, and p are either the same or different and independentlyeither 0 or 1 with the proviso that m, n, and p are not simultaneously0; q is 0, 1, 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.In one embodiment, R₁ and R₂ are each linoleyl, and the amino lipid is adilinoleyl amino lipid. In one embodiment, the amino lipid is adilinoleyl amino lipid.

In various other embodiments, a cationic lipid of Embodiments 1, 2 or 3(e.g., the cationic lipid, the first cationic lipid, the second cationiclipid) has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

R₁ and R₂ are independently selected from the group consisting of H, andC₁-C₃ alkyls;

R₃ and R₄ are independently selected from the group consisting of alkylgroups having from about 10 to about 20 carbon atoms, wherein at leastone of R₃ and R₄ comprises at least two sites of unsaturation. (e.g., R₃and R₄ may be, for example, dodecadienyl, tetradecadienyl,hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R₃and R₄ are both linoleyl. R₃ and R₄ may comprise at least three sites ofunsaturation (e.g., R₃ and R₄ may be, for example, dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl).

In some embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

R₁ and R₂ are independently selected and are H or C₁-C₃ alkyls. R₃ andR₄ are independently selected and are alkyl groups having from about 10to about 20 carbon atoms, wherein at least one of R₄ and R₄ comprises atleast two sites of unsaturation. In one embodiment, R₃ and R₄ are boththe same, for example, in some embodiments R₃ and R₄ are both linoleyl(i.e., C18), etc. In another embodiment, R₃ and R₄ are different, forexample, in some embodiments R₃ is tetradectrienyl (C14) and R₄ islinoleyl (C18). In a preferred embodiment, the cationic lipids) of thepresent invention are symmetrical, i.e., R₃ and R₄ are the same. Inanother preferred embodiment, both R₃ and R₄ comprise at least two sitesof unsaturation. In some embodiments, R₃ and R₄ are independentlyselected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl,and icosadienyl. In a preferred embodiment, R₃ and R₄ are both linoleyl.In some embodiments, R₄ and R₄ comprise at least three sites ofunsaturation and are independently selected from, e.g., dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

In various embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)has the formula:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

X_(aa) is a D- or L-amino acid residue having the formula—NR^(N)—CR¹R²—C(C═O)—, or a peptide or a peptide of amino acid residueshaving the formula —{NR^(N)—CR¹R²—(C═O)}_(n)—, wherein n is 2 to 20;

R¹ is independently, for each occurrence, a non-hydrogen, substituted orunsubstituted side chain of an amino acid;

R² and R^(N) are independently, for each occurrence, hydrogen, anorganic group consisting of carbon, oxygen, nitrogen, sulfur, andhydrogen atoms, or any combination of the foregoing, and having from 1to 20 carbon atoms, C₍₁₋₅₎alkyl, cycloalkyl, cycloalkylalkyl,C₍₃₋₅₎alkenyl, C₍₃₋₅₎alkynyl, C₍₁₋₅₎alkanoyl, C₍₁₋₅₎alkanoyloxy,C₍₁₋₅₎alkoxy, C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkyl, C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkoxy,C₍₁₋₅₎alkyl-amino-C₍₁₋₅₎alkyl-, C₍₁₋₅₎dialkyl-amino-C₍₁₋₅₎alkyl-,nitro-C₍₁₋₅₎alkyl, cyano-C₍₁₋₅₎alkyl, aryl-C₍₁₋₅₎alkyl,4-biphenyl-C₍₁₋₅₎alkyl, carboxyl, or hydroxyl;

Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker consisting of1-40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfuratoms (preferably, Z is NH or O);

R^(x) and R^(y) are, independently, (i) a lipophilic tail derived from alipid (which can be naturally-occurring or synthetic), phospholipid,glycolipid, triacylglycerol, glycerophospholipid, sphingolipid,ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tailoptionally includes a steroid; (ii) an amino acid terminal groupselected from hydrogen, hydroxyl, amino, and an organic protectinggroup; or (iii) a substituted or unsubstituted C₍₃₋₂₂₎alkyl,C₍₆₋₁₂₎cycloalkyl, C₍₆₋₁₂₎cycloalkyl-C₍₃₋₂₂₎alkyl, C₍₃₋₂₂₎alkenyl,C₍₃₋₂₂₎alkynyl, C₍₁₋₂₂₎alkoxy, or C₍₆₋₁₂₎-alkoxy-C₍₃₋₂₂₎alkyl;

one of R^(x) and R^(y) is a lipophilic tail as defined above and theother is an amino acid terminal group, or both R^(x) and R^(y) arelipophilic tails;

at least one of R^(x) and R^(y) is interrupted by one or morebiodegradable groups (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—,—C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—,—C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—,—OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)— or

wherein R¹¹ is a C₂-C₈ alkyl or alkenyl and each occurrence of R⁵ is,independently, H or alkyl; and each occurrence of R³ and R⁴ are,independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, ordialkylamino; or R³ and R⁴, together with the carbon atom to which theyare directly attached, form a cycloalkyl group (in one preferredembodiment, each occurrence of R³ and R⁴ are, independently H or C₁-C₄alkyl)); and R^(x) and R^(y) each, independently, optionally have one ormore carbon-carbon double bonds.

In some embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)is one of the following:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

R₁ and R₂ are independently alkyl, alkenyl or alkynyl, and each can beoptionally substituted;

R₃ and R₄ are independently a C₁-C₆ alkyl, or R₃ and R₄ can be takentogether to form an optionally substituted heterocyclic ring.

A representative useful dilinoleyl amino lipid has the formula:

wherein n is 0, 1, 2, 3, or 4.

In one embodiment, a cationic lipid of Embodiments 1, 2 or 3 (e.g., thecationic lipid, the first cationic lipid, the second cationic lipid) isDLin-K-DMA. In one embodiment, a cationic lipid of any one of thedisclosed embodiments (e.g., the cationic lipid, the first cationiclipid, the second cationic lipid) is DLin-KC2-DMA (DLin-K-DMA above,wherein n is 2).

In one embodiment, a cationic lipid of Embodiments 1, 2 or 3 (e.g., thecationic lipid, the first cationic lipid, the second cationic lipid) hasthe following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl or optionally substituted C₁₀-C₃₀acyl;

R₃ is H, optionally substituted C₁₀-C₁₀ alkyl, optionally substitutedalkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, ω-thiophosphoalkyl,optionally substituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K) heteroaryl, or heterocycle, orlinker-ligand, for example in some embodiments R₃ is (CH₃)₂N(CH₂)_(n)—,wherein n is 1, 2, 3 or 4;

E is O, S, N(Q), C(O), OC(O), C(O)O, N(Q)C(O), C(O)N(Q), (Q)N(CO)O,O(CO)N(Q), S(O), NS(O)₂N(Q), S(O)₂, N(Q)S(O)₂, SS, O═N, aryl,heteroaryl, cyclic or heterocycle, for example —C(O)O, wherein - is apoint of connection to R₃; and

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkylor ω-thiophosphoalkyl.

In one specific embodiment, a cationic lipid of Embodiments 1, 2 or 3(e.g., the cationic lipid, the first cationic lipid, the second cationiclipid) has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O),NS(O)₂N(Q), S(O)₂, N(Q)S(O)₂, SS, O═N, aryl, heteroaryl, cyclic orheterocycle;

Q is H, alkyl, ω-amninoalkyl, ω-(substituted)amninoalky, ω-phosphoalkylor ω-thiophosphoalkyl;

R₁ and R₂ and R_(x) are each independently for each occurrence H,optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₁₀-C₃₀alkyl, optionally substituted C₁₀-C₃₀ alkenyl, optionallysubstituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀ acyl, orlinker-ligand, provided that at least one of R₁, R₂ and R_(x) is not H;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, ω-thiophosphoalkyl,optionally substituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K) heteroaryl, or heterocycle, orlinker-ligand; and

n is 0, 1, 2, or 3.

In certain embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)has one of the following structures:

In one embodiment, a cationic lipid of Embodiments 1, 2 or 3 (e.g., thecationic lipid, the first cationic lipid, the second cationic lipid) hasthe structure of Formula I:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—,—(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a), —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond;

R^(a) is H or C₁-C₁₂ alkyl;

R^(1a) and R^(1b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(1b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(2a) and R^(2b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(3a) and R^(3b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(4a) and R^(4b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R⁵ and R⁶ are each independently methyl or cycloalkyl;

R⁷ is, at each occurrence, independently H or C₁-C₁₂ alkyl;

R⁸ and R⁹ are each independently unsubstituted C₁-C₁₂ alkyl; or R⁸ andR⁹, together with the nitrogen atom to which they are attached, form a5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

a and d are each independently an integer from 0 to 24;

b and c are each independently an integer from 1 to 24;

e is 1 or 2; and

x is 0, 1 or 2.

In some embodiments of Formula I, L¹ and L² are independently —O(C═O)—or —(C═O)O—.

In certain embodiments of Formula I, at least one of R^(1a), R^(2a),R^(3a) or R^(4a) is C₁-C₁₂ alkyl, or at least one of L¹ or L² is—O(C═O)— or —(C═O)O—. In other embodiments, R^(1a) and R^(1b) are notisopropyl when a is 6 or n-butyl when a is 8.

In still further embodiments of Formula I, at least one of R^(1a),R^(2a), R^(3a) or R^(4a) is C₁-C₁₂ alkyl, or at least one of L¹ or L² is—O(C═O)— or —(C═O)O—; and

R^(1a) and R^(1b) are not isopropyl when a is 6 or n-butyl when a is 8.

In other embodiments of Formula I, R⁸ and R⁹ are each independentlyunsubstituted C₁-C₁₂ alkyl; or R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring comprising one nitrogen atom;

In certain embodiments of Formula I, any one of L¹ or L² may be —O(C═O)—or a carbon-carbon double bond. L¹ and L² may each be —O(C═O)— or mayeach be a carbon-carbon double bond.

In some embodiments of Formula I, one of L¹ or L² is —O(C═O)—. In otherembodiments, both L¹ and L² are —O(C═O)—.

In some embodiments of Formula I, one of L¹ or L² is —(C═O)O—. In otherembodiments, both L¹ and L² are —(C═O)O—.

In some other embodiments of Formula I, one of L¹ or L² is acarbon-carbon double bond. In other embodiments, both L¹ and L² are acarbon-carbon double bond.

In still other embodiments of Formula I, one of L¹ or L² is —O(C═O)— andthe other of L¹ or L² is —(C═O)O—. In more embodiments, one of L¹ or L²is —O(C═O)— and the other of L¹ or L² is a carbon-carbon double bond. Inyet more embodiments, one of L¹ or L² is —(C═O)O— and the other of L¹ orL² is a carbon-carbon double bond.

It is understood that “carbon-carbon” double bond, as used throughoutthe specification, refers to one of the following structures:

wherein R^(a) and R^(b) are, at each occurrence, independently H or asubstituent. For example, in some embodiments R^(a) and R^(b) are, ateach occurrence, independently H, C₁-C₁₂ alkyl or cycloalkyl, forexample H or C₁-C₁₂ alkyl.

In other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ia):

In other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ib):

In yet other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ic):

In certain embodiments of the lipid compound of Formula I, a, b, c and dare each independently an integer from 2 to 12 or an integer from 4 to12. In other embodiments, a, b, c and d are each independently aninteger from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. Insome embodiments, a is 1. In other embodiments, a is 2. In moreembodiments, a is 3. In yet other embodiments, a is 4. In someembodiments, a is 5. In other embodiments, a is 6. In more embodiments,a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9.In other embodiments, a is 10. In more embodiments, a is 11. In yetother embodiments, a is 12. In some embodiments, a is 13. In otherembodiments, a is 14. In more embodiments, a is 15. In yet otherembodiments, a is 16.

In some other embodiments of Formula I, b is 1. In other embodiments, bis 2. In more embodiments, b is 3. In yet other embodiments, b is 4. Insome embodiments, b is 5. In other embodiments, b is 6. In moreembodiments, b is 7. In yet other embodiments, b is 8. In someembodiments, b is 9. In other embodiments, b is 10. In more embodiments,b is 11. In yet other embodiments, b is 12. In some embodiments, b is13. In other embodiments, b is 14. In more embodiments, b is 15. In yetother embodiments, b is 16.

In some more embodiments of Formula I, c is 1. In other embodiments, cis 2. In more embodiments, c is 3. In yet other embodiments, c is 4. Insome embodiments, c is 5. In other embodiments, c is 6. In moreembodiments, c is 7. In yet other embodiments, c is 8. In someembodiments, c is 9. In other embodiments, c is 10. In more embodiments,c is 11. In yet other embodiments, c is 12. In some embodiments, c is13. In other embodiments, c is 14. In more embodiments, c is 15. In yetother embodiments, c is 16.

In some certain other embodiments of Formula I, d is 0. In someembodiments, d is 1. In other embodiments, d is 2. In more embodiments,d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5.In other embodiments, d is 6. In more embodiments, d is 7. In yet otherembodiments, d is 8. In some embodiments, d is 9. In other embodiments,d is 10. In more embodiments, d is 11. In yet other embodiments, d is12. In some embodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some other various embodiments of Formula I, a and d are the same. Insome other embodiments, b and c are the same. In some other specificembodiments, a and d are the same and b and c are the same.

The sum of a and b and the sum of c and din Formula I are factors whichmay be varied to obtain a lipid of formula I having the desiredproperties. In one embodiment, a and b are chosen such that their sum isan integer ranging from 14 to 24. In other embodiments, c and d arechosen such that their sum is an integer ranging from 14 to 24. Infurther embodiment, the sum of a and b and the sum of c and d are thesame. For example, in some embodiments the sum of a and b and the sum ofc and d are both the same integer which may range from 14 to 24. Instill more embodiments, a, b, c and d are selected such the sum of a andb and the sum of c and d is 12 or greater.

In some embodiments of Formula I, e is 1. In other embodiments, e is 2.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) of Formula I arenot particularly limited. In certain embodiments R^(1a), R^(2a), R^(3a)and R^(4a) are H at each occurrence. In certain other embodiments atleast one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂ alkyl. Incertain other embodiments at least one of R^(1a), R^(2a), R^(3a) andR^(4a) is C₁-C₈ alkyl. In certain other embodiments at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula I, R^(1a), R^(1b), R^(4a) and R^(4b)are C₁-C₁₂ alkyl at each occurrence.

In further embodiments of Formula I, at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of Formula I, R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ of Formula I are not particularly limitedin the foregoing embodiments. In certain embodiments one or both of R⁵or R⁶ is methyl. In certain other embodiments one or both of R⁵ or R⁶ iscycloalkyl for example cyclohexyl. In these embodiments the cycloalkylmay be substituted or not substituted. In certain other embodiments thecycloalkyl is substituted with C₁-C₁₂ alkyl, for example tert-butyl.

The substituents at R⁷ are not particularly limited in the foregoingembodiments of Formula I. In certain embodiments at least one R⁷ is H.In some other embodiments, R⁷ is H at each occurrence. In certain otherembodiments R⁷ is C₁-C₁₂ alkyl.

In certain other of the foregoing embodiments of Formula I, one of R⁸ orR⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula I, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 5, 6 or 7-memberedheterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹,together with the nitrogen atom to which they are attached, form a5-membered heterocyclic ring, for example a pyrrolidinyl ring.

In some embodiments of Embodiment 3, the first and second cationiclipids are each, independently selected from a lipid of Formula I.

In various different embodiments, the lipid of Formula I has one of thestructures set forth in Table 1 below.

TABLE 1 Representative Lipids of Formula I Prep. No. Structure MethodpKa I-1

B — I-2

A 5.64 I-3

A 7.15 I-4

B 6.43 I-5

B 6.28 I-6

B 6.12 I-7

A — I-8

A — I-9

B — I-10

A — I-11

A 6.36 I-12

A — I-13

A 6.51 I-14

A — I-15

A 6.30 I-16

A 6.63 I-17

A — I-18

A — I-19

A 6.72 I-20

A 6.44 I-21

A 6.28 I-22

A 6.53 I-23

A 6.24 I-24

A 6.28 I-25

A 6.20 I-26

A 6.89 I-27

A 6.30 I-28

A 6.20 I-29

A 6.22 I-30

A — I-31

C 6.33 I-32

C 6.47 I-33

C 6.27 I-34

B — I-35

B 6.21 I-36

C — I-37

C — I-38

B 6.24 I-39

B 5.82 I-40

B 6.38 I-41

B 5.91

In some embodiments the lipid of Formula I is compound I-5. In someembodiments the lipid of Formula I is compound I-6.

In some embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)has a structure of Formula II:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—,—(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond;

G¹ is C₁-C₂ alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)— or adirect bond;

G² is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct bond;

G³ is C₁-C₆ alkylene;

R^(a) is H or C₁-C₁₂ alkyl;

R^(1a) and R^(1b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(1b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(2a) and R^(2b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(3a) and R^(3b) are, at each occurrence, independently either (a): Hor C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(4a) and R^(4b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R⁵ and R⁶ are each independently H or methyl;

R⁷ is C₄-C₂₀ alkyl;

R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹, togetherwith the nitrogen atom to which they are attached, form a 5, 6 or7-membered heterocyclic ring;

a, b, c and d are each independently an integer from 1 to 24; and

x is 0, 1 or 2.

In some embodiments of Formula (II), L¹ and L² are each independently—O(C═O)—, —(C═O)O— or a direct bond. In other embodiments, G¹ and G² areeach independently —(C═O)— or a direct bond. In some differentembodiments, L¹ and L² are each independently —O(C═O)—, —(C═O)O— or adirect bond; and G¹ and G² are each independently —(C═O)— or a directbond.

In some different embodiments of Formula (II), L¹ and L² are eachindependently —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a), —OC(═O)NR^(a)—,—NR^(a)C(═O)O—, —NR^(a)S(O)_(x)NR^(a)—, —NR^(a)S(O)_(x)— or—S(O)_(x)NR^(a)—.

In other of the foregoing embodiments of Formula (II), the lipidcompound has one of the following Formulae (IIA) or (IIB):

In some embodiments of Formula (II), the lipid compound has Formula(IIA). In other embodiments, the lipid compound has Formula (IIB).

In any of the foregoing embodiments of Formula (II), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—.

In some different embodiments of Formula (II), one of L¹ or L² is—(C═O)O—. For example, in some embodiments each of L¹ and L² is—(C═O)O—.

In different embodiments of Formula (II), one of L¹ or L² is a directbond. As used herein, a “direct bond” means the group (e.g., L¹ or L²)is absent. For example, in some embodiments each of L¹ and L² is adirect bond.

In other different embodiments of Formula (II), for at least oneoccurrence of R^(1a) and R^(1b), R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(1b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In still other different embodiments of Formula (II), for at least oneoccurrence of R^(4a) and R^(4b), R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(4b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In more embodiments of Formula (II), for at least one occurrence ofR^(2a) and R^(2b), R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together withthe carbon atom to which it is bound is taken together with an adjacentR^(2b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

In other different embodiments of Formula (II), for at least oneoccurrence of R^(3a) and R^(3b), R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(3b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In various other embodiments of Formula (II), the lipid compound has oneof the following Formulae (IIC) or (IID):

wherein e, f, g and h are each independently an integer from 1 to 12.

In some embodiments of Formula (II), the lipid compound has Formula(IIC). In other embodiments, the lipid compound has Formula (IID).

In various embodiments of Formulae (IIC) or (IID), e, f, g and h areeach independently an integer from 4 to 10.

In certain embodiments of Formula (II), a, b, c and d are eachindependently an integer from 2 to 12 or an integer from 4 to 12. Inother embodiments, a, b, c and d are each independently an integer from8 to 12 or 5 to 9. In some certain embodiments, a is 0. In someembodiments, a is 1. In other embodiments, a is 2. In more embodiments,a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5.In other embodiments, a is 6. In more embodiments, a is 7. In yet otherembodiments, a is 8. In some embodiments, a is 9. In other embodiments,a is 10. In more embodiments, a is 11. In yet other embodiments, a is12. In some embodiments, a is 13. In other embodiments, a is 14. In moreembodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments of Formula (II), b is 1. In other embodiments, b is2. In more embodiments, b is 3. In yet other embodiments, b is 4. Insome embodiments, b is 5. In other embodiments, b is 6. In moreembodiments, b is 7. In yet other embodiments, b is 8. In someembodiments, b is 9. In other embodiments, b is 10. In more embodiments,b is 11. In yet other embodiments, b is 12. In some embodiments, b is13. In other embodiments, b is 14. In more embodiments, b is 15. In yetother embodiments, b is 16.

In some embodiments of Formula (II), c is 1. In other embodiments, c is2. In more embodiments, c is 3. In yet other embodiments, c is 4. Insome embodiments, c is 5. In other embodiments, c is 6. In moreembodiments, c is 7. In yet other embodiments, c is 8. In someembodiments, c is 9. In other embodiments, c is 10. In more embodiments,c is 11. In yet other embodiments, c is 12. In some embodiments, c is13. In other embodiments, c is 14. In more embodiments, c is 15. In yetother embodiments, c is 16.

In some certain embodiments of Formula (II), d is 0. In someembodiments, d is 1. In other embodiments, d is 2. In more embodiments,d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5.In other embodiments, d is 6. In more embodiments, d is 7. In yet otherembodiments, d is 8. In some embodiments, d is 9. In other embodiments,d is 10. In more embodiments, d is 11. In yet other embodiments, d is12. In some embodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some embodiments of Formula (II), e is 1. In other embodiments, e is2. In more embodiments, e is 3. In yet other embodiments, e is 4. Insome embodiments, e is 5. In other embodiments, e is 6. In moreembodiments, e is 7. In yet other embodiments, e is 8. In someembodiments, e is 9. In other embodiments, e is 10. In more embodiments,e is 11. In yet other embodiments, e is 12.

In some embodiments of Formula (II), f is 1. In other embodiments, f is2. In more embodiments, f is 3. In yet other embodiments, f is 4. Insome embodiments, f is 5. In other embodiments, f is 6. In moreembodiments, f is 7. In yet other embodiments, f is 8. In someembodiments, f is 9. In other embodiments, f is 10. In more embodiments,f is 11. In yet other embodiments, f is 12.

In some embodiments of Formula (II), g is 1. In other embodiments, g is2. In more embodiments, g is 3. In yet other embodiments, g is 4. Insome embodiments, g is 5. In other embodiments, g is 6. In moreembodiments, g is 7. In yet other embodiments, g is 8. In someembodiments, g is 9. In other embodiments, g is 10. In more embodiments,g is 11. In yet other embodiments, g is 12.

In some embodiments of Formula (II), his 1. In other embodiments, e is2. In more embodiments, h is 3. In yet other embodiments, h is 4. Insome embodiments, e is 5. In other embodiments, h is 6. In moreembodiments, h is 7. In yet other embodiments, h is 8. In someembodiments, h is 9. In other embodiments, h is 10. In more embodiments,h is 11. In yet other embodiments, h is 12.

In some other various embodiments of Formula (II), a and d are the same.In some other embodiments, b and c are the same. In some other specificembodiments and a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d of Formula (II) are factorswhich may be varied to obtain a lipid having the desired properties. Inone embodiment, a and b are chosen such that their sum is an integerranging from 14 to 24. In other embodiments, c and d are chosen suchthat their sum is an integer ranging from 14 to 24. In furtherembodiment, the sum of a and b and the sum of c and d are the same. Forexample, in some embodiments the sum of a and b and the sum of c and dare both the same integer which may range from 14 to 24. In still moreembodiments, a, b, c and d are selected such that the sum of a and b andthe sum of c and d is 12 or greater.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) of Formula (II)are not particularly limited. In some embodiments, at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is H. In certain embodiments R^(1a),R^(2a), R^(3a) and R^(4a) are H at each occurrence. In certain otherembodiments at least one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂alkyl. In certain other embodiments at least one of R^(1a), R^(2a),R^(3a) and R^(4a) is C₁-C₈ alkyl. In certain other embodiments at leastone of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula (II), R^(1a), R^(1b), R^(4a) andR^(4b) are C₁-C₁₂ alkyl at each occurrence.

In further embodiments of Formula (II), at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of Formula (II), R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ of Formula (II) are not particularlylimited in the foregoing embodiments. In certain embodiments one of R⁵or R⁶ is methyl. In other embodiments each of R⁵ or R⁶ is methyl.

The substituents at R⁷ of Formula (II) are not particularly limited inthe foregoing embodiments. In certain embodiments R⁷ is C₆-C₁₆ alkyl. Insome other embodiments, R⁷ is C₆-C₉ alkyl. In some of these embodiments,R⁷ is substituted with —(C═O)OR^(b), —O(C═O)R^(b), —C(═O)R^(b), —OR^(b),—S(O)_(x)R^(b), —S—SR^(b), —C(═O)SR^(b), —SC(═O)R^(b), —NR^(a)R^(b),—NR^(a)C(═O)R^(b), —C(═O)NR^(a)R^(b), —NR^(a)C(═O)NR^(a)R^(b),—OC(═O)NR^(a)R^(b), —NR^(a)C(═O)OR^(b), —NR^(a)S(O)_(x)NR^(a)R^(b),—NR^(a)S(O)_(x)R^(b) or —S(O)_(x)NR^(a)R^(b), wherein: R^(a) is H orC₁-C₁₂ alkyl; R^(b) is C₁-C₁₅ alkyl; and x is 0, 1 or 2. For example, insome embodiments R⁷ is substituted with —(C═O)OR^(b) or —O(C═O)R^(b).

In some of the foregoing embodiments of Formula (II), R^(b) is branchedC₁-C₁₆ alkyl. For example, in some embodiments R^(b) has one of thefollowing structures:

In certain other of the foregoing embodiments of Formula (II), one of R⁸or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula (II), R⁸ and R⁹, together withthe nitrogen atom to which they are attached, form a 5, 6 or 7-memberedheterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹,together with the nitrogen atom to which they are attached, form a5-membered heterocyclic ring, for example a pyrrolidinyl ring. In somedifferent embodiments of the foregoing, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 6-membered heterocyclicring, for example a piperazinyl ring.

In certain embodiments of Embodiment 3, the first and second cationiclipids are each, independently selected from a lipid of Formula II.

In still other embodiments of the foregoing lipids of Formula (II), G³is C₂-C₄ alkylene, for example C₃ alkylene. In various differentembodiments, the lipid compound has one of the structures set forth inTable 2 below

TABLE 2 Representative Lipids of Formula (II) Prep. No. Structure MethodpKa II-1

D 5.64 II-2

D — II-3

D — II-4

E — II-5

D 6.27 II-6

D 6.14 II-7

D 5.93 II-8

D 5.35 II-9

D 6.27 II-10

D 6.16 II-11

D 6.13 II-12

D 6.21 II-13

D 6.22 II-14

D 6.33 II-15

D 6.32 II-16

E 6.37 II-17

F 6.27 II-18

D — II-19

D — II-20

D — II-21

D — II-22

D — II-23

D — II-24

D 6.14 II-25

E — II-26

E — II-27

E — II-28

E — II-29

E — II-30

E — II-31

E — II-32

E — II-33

E — II-34

E — II-35

D 5.97 II-36

F 6.13 II-37

D 5.61 II-38

D 6.45 II-39

D 6.45 II-40

D 6.57 II-41

D — II-42

D — II-43

F — II-44

D — II-45

D — II-46

D —

In some embodiments the lipid of Formula (II) is compound II-9. In someembodiments the lipid of Formula (II) is compound II-10. In someembodiments the lipid of Formula (II) is compound II-11. In someembodiments the lipid of Formula (II) is compound II-12. In someembodiments the lipid of Formula (II) is compound II-14. In someembodiments the lipid of Formula (II) is compound II-15.

In some other embodiments, a cationic lipid of Embodiments 1, 2 or 3(e.g., the cationic lipid, the first cationic lipid, the second cationiclipid) has a structure of Formula III:

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—,—(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond;

G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂alkenylene;

G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈cycloalkenylene;

R^(a) is H or C₁-C₁₂ alkyl;

R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;

R³ is H, OR^(S), CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;

R⁴ is C₁-C₁₂ alkyl;

R⁵ is H or C₁-C₆ alkyl; and

x is 0, 1 or 2.

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following Formulae (IIIA) or (IIIB):

wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;

n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of Formula (III), the lipid hasFormula (IIIA), and in other embodiments, the lipid has Formula (IIIB).

In other embodiments of Formula (III), the lipid has one of thefollowing Formulae (IIIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments of Formula (III), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—. In some different embodiments of any of the foregoing, L¹ andL² are each independently —(C═O)O— or —O(C═O)—. For example, in someembodiments each of L¹ and L² is —(C═O)O—.

In some different embodiments of Formula (III), the lipid has one of thefollowing Formulae (IIIE) or (IIIF):

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following Formulae (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integerranging from 2 to 12, for example from 2 to 8 or from 2 to 4. Forexample, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5. In someembodiments, n is 6.

In some other of the foregoing embodiments of Formula (III), y and z areeach independently an integer ranging from 2 to 10. For example, in someembodiments, y and z are each independently an integer ranging from 4 to9 or from 4 to 6.

In some of the foregoing embodiments of Formula (III), R⁶ is H. In otherof the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments,R⁶ is OH.

In some embodiments of Formula (III), G³ is unsubstituted. In otherembodiments, G3 is substituted. In various different embodiments, G³ islinear C₁-C₂₄ alkylene or linear C₁-C₂₄ alkenylene.

In some other foregoing embodiments of Formula (III), R¹ or R², or both,is C₆-C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each,independently have the following structure:

wherein:

R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂alkyl; and

a is an integer from 2 to 12,

wherein R^(7a), R^(7b) and a are each selected such that R¹ and R² eachindependently comprise from 6 to 20 carbon atoms. For example, in someembodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments of Formula (III), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of Formula (III), R¹ or R², or both, has one ofthe following structures:

In some of the foregoing embodiments of Formula (III), R³ is OH, CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl orethyl.

In some specific embodiments of Embodiment 3, the first and secondcationic lipids are each, independently selected from a lipid of FormulaIII.

In various different embodiments, a cationic lipid of any one of thedisclosed embodiments (e.g., the cationic lipid, the first cationiclipid, the second cationic lipid) of Formula (III) has one of thestructures set forth in Table 3 below.

TABLE 3 Representative Compounds of Formula (III) Prep. No. StructureMethod pKa III-1

G 5.89 III-2

G 6.05 III-3

G 6.09 III-4

G 5.60 III-5

G 5.59 III-6

G 5.42 III-7

G 6.11 III-8

G 5.84 III-9

G — III-10

G — III-11

G — III-12

G — III-13

G — III-14

G — III-15

G 6.14 III-16

G 6.31 III-17

G 6.28 III-18

G — III-19

G — III-20

G 6.36 III-21

G — III-22

G 6.10 III-23

G 5.98 III-24

G — III-25

G 6.22 III-26

G 5.84 III-27

G 5.77 III-28

G — III-29

G — III-30

G 6.09 III-31

G — III-32

G — III-33

G — III-34

G — III-35

G — III-36

G — III-37

G — III-38

G — III-39

G — III-40

G — III-41

G — III-42

G — III-43

G — III-44

G — III-45

G — III-46

G — III-47

G — III-48

G — III-49

G —

In some embodiments the lipid of Formula (III) is compound III-3. Insome embodiments the lipid of Formula (III) is compound III-25.

In one embodiment, the first cationic lipid, or the second cationiclipid, or both, independently has a structure of Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—,—O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond;

L is, at each occurrence, —O(C═O)—, wherein - represents a covalent bondto X;

X is CR^(a);

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least onepolar functional group when n is 1; or Z is alkylene, cycloalkylene or apolyvalent moiety comprising at least one polar functional group when nis greater than 1;

R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl, C₁-C₁₂hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl;

R is, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl;or (b) R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond;

R¹ and R² have, at each occurrence, the following structure,respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to12;

b¹ and b² are, at each occurrence, independently 0 or 1;

c¹ and c² are, at each occurrence, independently an integer from 5 to10;

d¹ and d² are, at each occurrence, independently an integer from 5 to10;

y is, at each occurrence, independently an integer from 0 to 2; and

n is an integer from 1 to 6,

wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl,alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted withone or more substituent.

In some embodiments of Formula (IV), G¹ and G² are each independently—O(C═O)— or —(C═O)O—.

In other embodiments of Formula (IV), X is CH.

In different embodiments of Formula (IV), the sum of a¹+b¹+c¹ or the sumof a²+b²+c² is an integer from 12 to 26.

In still other embodiments of Formula (IV), a¹ and a² are independentlyan integer from 3 to 10. For example, in some embodiments a¹ and a² areindependently an integer from 4 to 9.

In various embodiments of Formula (IV), b¹ and b² are 0. In differentembodiments, b¹ and b² are 1.

In more embodiments of Formula (IV), c¹, c², d¹ and d² are independentlyan integer from 6 to 8.

In other embodiments of Formula (IV), c¹ and c² are, at each occurrence,independently an integer from 6 to 10, and d¹ and d² are, at eachoccurrence, independently an integer from 6 to 10.

In other embodiments of Formula (IV), c¹ and c² are, at each occurrence,independently an integer from 5 to 9, and d¹ and d² are, at eachoccurrence, independently an integer from 5 to 9.

In more embodiments of Formula (IV), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In various embodiments of the foregoing Formula (IV), R is, at eachoccurrence, independently either: (a) H or methyl; or (b) R togetherwith the carbon atom to which it is bound is taken together with anadjacent R and the carbon atom to which it is bound to form acarbon-carbon double bond. In certain embodiments, each R is H. In otherembodiments at least one R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond.

In other embodiments of the compound of Formula (IV), R¹ and R²independently have one of the following structures:

In certain embodiments of Formula (IV), the compound has one of thefollowing structures:

In still different embodiments a cationic lipid of Embodiments 1, 2 or 3(e.g., the cationic lipid, the first cationic lipid, the second cationiclipid) has the structure of Formula (V):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—,—O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond;

L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a covalent bondto X;

X is CR^(a);

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least onepolar functional group when n is 1; or Z is alkylene, cycloalkylene or apolyvalent moiety comprising at least one polar functional group when nis greater than 1;

R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl, C₁-C₁₂hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl;

R is, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl;or (b) R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond;

R¹ and R² have, at each occurrence, the following structure,respectively:

R′ is, at each occurrence, independently H or C₁-C₁₂ alkyl;

a¹ and a² are, at each occurrence, independently an integer from 3 to12;

b¹ and b² are, at each occurrence, independently 0 or 1;

c¹ and c² are, at each occurrence, independently an integer from 2 to12;

d¹ and d² are, at each occurrence, independently an integer from 2 to12;

y is, at each occurrence, independently an integer from 0 to 2; and

n is an integer from 1 to 6,

wherein a¹, a², c¹, c², d¹ and d² are selected such that the sum ofa¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d² is aninteger from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl,aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl,alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionallysubstituted with one or more substituent.

In certain embodiments of Formula (V), G¹ and G² are each independently—O(C═O)— or —(C═O)O—.

In other embodiments of Formula (V), X is CH.

In some embodiments of Formula (V), the sum of a¹+c¹+d¹ is an integerfrom 20 to 30, and the sum of a²+c²+d² is an integer from 18 to 30. Inother embodiments, the sum of a¹+c¹+d¹ is an integer from 20 to 30, andthe sum of a²+c²+d² is an integer from 20 to 30. In more embodiments ofFormula (V), the sum of a¹+b¹+c¹ or the sum of a²+b²+c² is an integerfrom 12 to 26. In other embodiments, a¹, a², c¹, c², d¹ and d² areselected such that the sum of a¹+c¹+d¹ is an integer from 18 to 28, andthe sum of a²+c²+d² is an integer from 18 to 28,

In still other embodiments of Formula (V), a¹ and a² are independentlyan integer from 3 to 10, for example an integer from 4 to 9.

In yet other embodiments of Formula (V), b¹ and b² are 0. In differentembodiments b¹ and b² are 1.

In certain other embodiments of Formula (V), c¹, c², d¹ and d² areindependently an integer from 6 to 8.

In different other embodiments of Formula (V), Z is alkyl or amonovalent moiety comprising at least one polar functional group when nis 1; or Z is alkylene or a polyvalent moiety comprising at least onepolar functional group when n is greater than 1.

In more embodiments of Formula (V), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In other different embodiments of Formula (V), R is, at each occurrence,independently either: (a) H or methyl; or (b) R together with the carbonatom to which it is bound is taken together with an adjacent R and thecarbon atom to which it is bound to form a carbon-carbon double bond.For example in some embodiments each R is H. In other embodiments atleast one R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond.

In more embodiments, each R′ is H.

In certain embodiments of Formula (V), the sum of a¹+c¹+d¹ is an integerfrom 20 to 25, and the sum of a²+c²+d² is an integer from 20 to 25.

In other embodiments of Formula (V), R¹ and R² independently have one ofthe following structures:

In more embodiments of Formula (V), the compound has one of thefollowing structures:

In any of the foregoing embodiments of Formula (IV) or (V), n is 1. Inother of the foregoing embodiments of Formula (IV) or (V), n is greaterthan 1.

In more of any of the foregoing embodiments of Formula (IV) or (V), Z isa mono- or polyvalent moiety comprising at least one polar functionalgroup. In some embodiments, Z is a monovalent moiety comprising at leastone polar functional group. In other embodiments, Z is a polyvalentmoiety comprising at least one polar functional group.

In more of any of the foregoing embodiments of Formula (IV) or (V), thepolar functional group is a hydroxyl, alkoxy, ester, cyano, amide,amino, alkylaminyl, heterocyclyl or heteroaryl functional group.

In any of the foregoing embodiments of Formula (IV) or (V), Z ishydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl,alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.

In some other embodiments of Formula (IV) or (V), Z has the followingstructure:

wherein:

R⁵ and R⁶ are independently H or C₁-C₆ alkyl;

R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸, together withthe nitrogen atom to which they are attached, join to form a 3-7membered heterocyclic ring; and

x is an integer from 0 to 6.

In still different embodiments of Formula (IV) or (V), Z has thefollowing structure:

wherein:

R⁵ and R⁶ are independently H or C₁-C₆ alkyl;

R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸, together withthe nitrogen atom to which they are attached, join to form a 3-7membered heterocyclic ring; and

x is an integer from 0 to 6.

In still different embodiments of formula (IV) or (V), Z has thefollowing structure:

wherein:

R⁵ and R⁶ are independently H or C₁-C₆ alkyl;

R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸, together withthe nitrogen atom to which they are attached, join to form a 3-7membered heterocyclic ring; and

x is an integer from 0 to 6.

In some other embodiments of Formula (IV) or (V), Z is hydroxylalkyl,cyanoalkyl or an alkyl substituted with one or more ester or amidegroups.

For example, in any of the foregoing embodiments of Formula (IV) or (V),Z has one of the following structures:

In other embodiments of Formula (IV) or (V), Z-L has one of thefollowing structures:

In other embodiments, Z-L has one of the following structures:

In still other embodiments, X is CH and Z-L has one of the followingstructures:

In some specific embodiments of Embodiment 3, the first and secondcationic lipids are each, independently selected from a lipid of FormulaIV. In some embodiments of Embodiment 3, the first and second cationiclipids are each, independently selected from a lipid of Formula V.

In various different embodiments, a cationic lipid of any one of thedisclosed embodiments (e.g., the cationic lipid, the first cationiclipid, the second cationic lipid) has one of the structures set forth inTable 4 below.

TABLE 4 Representative Compounds of Formula (IV) or (V) No. StructureIV-1

IV-2

IV-3

In certain embodiments, the neutral lipid is present in any of theforegoing LNPs in a concentration ranging from 5 to 10 mol percent, from5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. Incertain specific embodiments, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent. In some embodiments,the molar ratio of cationic lipid to the neutral lipid ranges from about4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or fromabout 4.7:1.0 to 4.8:1.0. In some embodiments, the molar ratio of totalcationic lipid to the neutral lipid ranges from about 4.1:1.0 to about4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to4.8:1.0.

In some embodiments, a cationic lipid of Embodiments 1, 2 or 3 (e.g.,the cationic lipid, the first cationic lipid, the second cationic lipid)has one of the following structures:

In some embodiments of Embodiments 1, 2 or 3, the cationic lipid has thefollowing structure:

In other embodiments of Embodiment 3, the first and second cationiclipids have the following structures, respectively:

Exemplary neutral lipids for use in any of Embodiments 1, 2 or 3include, for example, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). In some embodiments,the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPEand SM. In some embodiments, the neutral lipid is DSPC.

In various embodiments, any of the disclosed lipid nanoparticlescomprise a steroid or steroid analogue. In certain embodiments, thesteroid or steroid analogue is cholesterol. In some embodiments, thesteroid is present in a concentration ranging from 39 to 49 molarpercent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molarpercent. In certain specific embodiments, the steroid is present in aconcentration of 40, 41, 42, 43, 44, 45, or 46 molar percent.

In certain embodiments, the molar ratio of cationic lipid to the steroidranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some ofthese embodiments, the molar ratio of cationic lipid to cholesterolranges from about 5:1 to 1:1. In certain embodiments, the steroid ispresent in a concentration ranging from 32 to 40 mol percent of thesteroid.

In certain embodiments, the molar ratio of total cationic lipid (i.e.,the sum of the first and second cationic lipid) to the steroid rangesfrom 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some of theseembodiments, the molar ratio of total cationic lipid to cholesterolranges from about 5:1 to 1:1. In certain embodiments, the steroid ispresent in a concentration ranging from 32 to 40 mol percent of thesteroid.

In various other embodiments of Embodiments 1, 2 or 3, the polymerconjugated lipid is a pegylated lipid. For example, some embodimentsinclude a pegylated diacylglycerol (PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

In various embodiments, the polymer conjugated lipid is present in aconcentration ranging from 1.0 to 2.5 molar percent. In certain specificembodiments, the polymer conjugated lipid is present in a concentrationof about 1.7 molar percent. In some embodiments, the polymer conjugatedlipid is present in a concentration of about 1.5 molar percent.

In certain embodiments, the molar ratio of cationic lipid to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In someembodiments, the molar ratio of cationic lipid to polymer conjugatedlipid ranges from about 100:1 to about 20:1.

In certain embodiments, the molar ratio of total cationic lipid (i.e.,the sum of the first and second cationic lipid) to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In someembodiments, the molar ratio of total cationic lipid to polymerconjugated lipid ranges from about 100:1 to about 20:1.

In some embodiments of Embodiments 1, 2 or 3, the pegylated lipid hasthe following Formula (VI):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R¹² and R¹³ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R¹² and R¹³ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w ranges from 42 to 55, for example, theaverage w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55.In some specific embodiments, the average w is about 49.

In some embodiments, the pegylated lipid has the following Formula(VIa):

wherein the average w is about 49.

In some embodiments of Embodiments 1, 2 or 3, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense and messenger RNA. For example,messenger RNA may be used to induce an immune response (e.g., as avaccine), for example by translation of immunogenic proteins.

In some embodiments, a plurality of the lipid nanoparticles has apolydispersity of less than 0.12, or less than 0.08. In someembodiments, the lipid nanoparticle has a mean diameter ranging from 50nm to 100 nm, or from 60 nm to 85 nm.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoing LNPsand/or administering a composition comprising the same to the patient.In some embodiments, the therapeutic agent is effective to treat thedisease.

For the purposes of administration, the lipid nanoparticles ofembodiments of the present invention may be administered alone or may beformulated as pharmaceutical compositions. Pharmaceutical compositionsof certain embodiments comprise a lipid nanoparticle according to any ofthe foregoing embodiments and one or more pharmaceutically acceptablecarrier, diluent or excipient. The lipid nanoparticle may be present inan amount which is effective to deliver the therapeutic agent, e.g., fortreating a particular disease or condition of interest. Appropriateconcentrations and dosages can be readily determined by one skilled inthe art.

Administration of the lipid nanoparticles of some embodiments can becarried out via any of the accepted modes of administration of agentsfor serving similar utilities. The pharmaceutical compositions of someembodiments may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suspensions, suppositories, injections, inhalants,gels, microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of some embodiments are formulated so as to allow theactive ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that may beadministered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container comprising LNPs in aerosol form may hold a plurality ofdosage units. Actual methods of preparing such dosage forms are known,or will be apparent, to those skilled in this art; for example, seeRemington: The Science and Practice of Pharmacy, 20th Edition(Philadelphia College of Pharmacy and Science, 2000). The composition tobe administered will typically contain a therapeutically effectiveamount of a lipid nanoparticle of any of the embodiments disclosedherein, comprising a therapeutic agent, or a pharmaceutically acceptablesalt thereof, for treatment of a disease or condition of interest.

A pharmaceutical composition of some embodiments may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of some embodiments, whether theybe solutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose; agents to act as cryoprotectants such assucrose or trehalose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of certain embodiments intended foreither parenteral or oral administration should contain an amount of alipid nanoparticle of the invention such that a suitable dosage will beobtained.

The pharmaceutical composition of embodiments of the invention may beintended for topical administration, in which case the carrier maysuitably comprise a solution, emulsion, ointment or gel base. The base,for example, may comprise one or more of the following: petrolatum,lanolin, polyethylene glycols, bee wax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers. Thickening agentsmay be present in a pharmaceutical composition for topicaladministration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device.

The pharmaceutical composition of some embodiments may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of other embodiments may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of embodiments in solid or liquid formmay include an agent that binds to the LNP or therapeutic agent, andthereby assists in the delivery of the LNP or therapeutic agent.Suitable agents that may act in this capacity include a monoclonal orpolyclonal antibody, or a protein.

In other embodiments, the pharmaceutical composition may comprise orconsist of dosage units that can be administered as an aerosol. The termaerosol is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemthat dispenses the active ingredients. Aerosols of compounds of theinvention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One skilledin the art, without undue experimentation may determine preferredaerosols.

In some embodiments, the pharmaceutical compositions may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining the lipid nanoparticles of the invention withsterile, distilled water or other carrier so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the compound of the invention so as to facilitatedissolution or homogeneous suspension of the compound in the aqueousdelivery system.

The pharmaceutical compositions of some embodiments are administered ina therapeutically effective amount, which will vary depending upon avariety of factors including the activity of the specific therapeuticagent employed; the metabolic stability and length of action of thetherapeutic agent; the age, body weight, general health, sex, and dietof the patient; the mode and time of administration; the rate ofexcretion; the drug combination; the severity of the particular disorderor condition; and the subject undergoing therapy.

The pharmaceutical compositions of various embodiments may also beadministered simultaneously with, prior to, or after administration ofone or more other therapeutic agents. Such combination therapy includesadministration of a single pharmaceutical dosage formulation of acomposition of the invention and one or more additional active agents,as well as administration of the composition of the invention and eachactive agent in its own separate pharmaceutical dosage formulation. Forexample, a pharmaceutical composition of one embodiments and the otheractive agent can be administered to the patient together in a singleoral dosage composition such as a tablet or capsule, or each agentadministered in separate oral dosage formulations. Where separate dosageformulations are used, the compounds of the invention and one or moreadditional active agents can be administered at essentially the sametime, i.e., concurrently, or at separately staggered times, i.e.,sequentially; combination therapy is understood to include all theseregimens.

Preparation methods for the above lipids, lipid nanoparticles andcompositions are described herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all lipids which exist in free base or acid form can beconverted to their pharmaceutically acceptable salts by treatment withthe appropriate inorganic or organic base or acid by methods known toone skilled in the art. Salts of the lipids can be converted to theirfree base or acid form by standard techniques.

The following Reaction Schemes illustrate methods to make lipids ofFormula (I), (II), (III), (IV) or (V).

Embodiments of the lipid of Formula (I) (e.g., compound A-5) can beprepared according to General Reaction Scheme 1 (“Method A”), wherein Ris a saturated or unsaturated C₁-C₂₄ alkyl or saturated or unsaturatedcycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring toGeneral Reaction Scheme 1, compounds of structure A-1 can be purchasedfrom commercial sources or prepared according to methods familiar to oneof ordinary skill in the art. A mixture of A-1, A-2 and DMAP is treatedwith DCC to give the bromide A-3. A mixture of the bromide A-3, a base(e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 isheated at a temperature and time sufficient to produce A-5 after anynecessarily workup and or purification step.

Other embodiments of the compound of Formula (I) (e.g., compound B-5)can be prepared according to General Reaction Scheme 2 (“Method B”),wherein R is a saturated or unsaturated C₁-C₂₄ alkyl or saturated orunsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Asshown in General Reaction Scheme 2, compounds of structure B-1 can bepurchased from commercial sources or prepared according to methodsfamiliar to one of ordinary skill in the art. A solution of B-1 (1equivalent) is treated with acid chloride B-2 (1 equivalent) and a base(e.g., triethylamine). The crude product is treated with an oxidizingagent (e.g., pyridinum chlorochromate) and intermediate product B-3 isrecovered. A solution of crude B-3, an acid (e.g., acetic acid), andN,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g.,sodium triacetoxyborohydride) to obtain B-5 after any necessary work upand/or purification.

It should be noted that although starting materials A-1 and B-1 aredepicted above as including only saturated methylene carbons, startingmaterials which include carbon-carbon double bonds may also be employedfor preparation of compounds which include carbon-carbon double bonds.

Different embodiments of the lipid of Formula (I) (e.g., compound C-7 orC9) can be prepared according to General Reaction Scheme 3 (“Method C”),wherein R is a saturated or unsaturated C₁-C₂₄ alkyl or saturated orunsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24.Referring to General Reaction Scheme 3, compounds of structure C-1 canbe purchased from commercial sources or prepared according to methodsfamiliar to one of ordinary skill in the art.

Embodiments of the compound of Formula (II) (e.g., compounds D-5 andD-7) can be prepared according to General Reaction Scheme 4 (“MethodD”), wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a),R^(4b), R⁵, R⁶, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as definedherein, and R^(7′) represents R⁷ or a C₃-C₁₉ alkyl. Referring to GeneralReaction Scheme 1, compounds of structure D-1 and D-2 can be purchasedfrom commercial sources or prepared according to methods familiar to oneof ordinary skill in the art. A solution of D-1 and D-2 is treated witha reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3after any necessary work up. A solution of D-3 and a base (e.g.,trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylicacid and DCC) to obtain D-5 after any necessary work up and/orpurification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after anynecessary work up and/or purification.

Embodiments of the lipid of Formula (II) (e.g., compound E-5) can beprepared according to General Reaction Scheme 5 (“Method E”), whereinR^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b), R⁵, R⁶,R⁷, R⁸, R⁹, L¹, L², G³, a, b, c and d are as defined herein. Referringto General Reaction Scheme 2, compounds of structure E-1 and E-2 can bepurchased from commercial sources or prepared according to methodsfamiliar to one of ordinary skill in the art. A mixture of E-1 (inexcess), E-2 and a base (e.g., potassium carbonate) is heated to obtainE-3 after any necessary work up. A solution of E-3 and a base (e.g.,trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylicacid and DCC) to obtain E-5 after any necessary work up and/orpurification.

Other embodiments of the compound of Formula (II) (e.g., F-9) areprepared according to General Reaction Scheme 6. As illustrated inGeneral Reaction Scheme 6, an appropriately protected ketone (F-1) isreacted under reductive amination conditions with amine F-2 to yieldF-3. Acylation of F-3 with acid chloride F-4 yields acylated productF-5. Removal of the alcohol protecting group on F-5 followed by reactionwith F-7 and/or F-8 and appropriate activating reagent (e.g., DCC)yields the desired compound F-9.

General Reaction Scheme 7 provides an exemplary method (Method G) forpreparation of Lipids of Formula (III), wherein L¹ and L² are —(C═O)O—.G¹, G³, R¹ and R³ in General Reaction Scheme 7 are as defined herein forFormula (III), and G^(1′) refers to a one-carbon shorter homologue ofG¹. Compounds of structure G-1 are purchased or prepared according tomethods known in the art. Reaction of G-1 with diol G-2 underappropriate condensation conditions (e.g., DCC) yields ester/alcoholG-3, which can then be oxidized (e.g., PCC) to aldehyde G-4. Reaction ofG-4 with amine G-5 under reductive amination conditions yields a lipidof Formula (III) , wherein L¹ and L² are —(C═O)O—.

General Reaction Schemes 8-10 provide exemplary methods for preparationof compounds of Formula (IV) or (V).

General Reaction Scheme 8 (“Method H”) provides a method for preparationof exemplary compounds of Formula (V) or (IV) (i.e., compound “H-7”),wherein R, R¹, a¹, a² and Z are as defined herein, and PG is an alcoholprotecting group such as tetrahydropyran. Compounds of structure H-1 arepurchased or prepared according to methods known in the art. Reaction ofH-1 with ethyl formate H-2 under Grignard conditions yields alcohol H-3,which can then be coupled with acid H-4 under standard conditions toyield H-5. Removal of the protecting group followed by coupling withacid H-6 yields H-7.

General Reaction Scheme 9 (“Method I”) provides an alternative methodfor preparation of exemplary compounds of Formula (V) or (IV) (i.e.,compound “I-9”), wherein R, R¹, a¹, a² and Z are as defined herein andPG is an alcohol protecting group such as tetrahydropyran. Compounds ofstructure I-1 are purchased or prepared according to methods known inthe art. The hydroxyl group of Compound I-1 is protected using methodsknown in the art (e.g., pyridinium p-toluenesulfonate, dihydropyran) toyield I-2. Reaction of I-2 with ethyl formate I-3 under Grignardconditions (e.g., with Mg, I₂) affords the alcohol I-4. The hydroxylgroup of compound I-4 can be oxidized (e.g., with pyridiniumchlorochromate) and the protecting groups removed (e.g., with pyridiniump-toluenesulfonate) to yield compound I-5. The free hydroxyl groups ofI-5 are then coupled with acid I-6 under standard ester couplingconditions to yield I-7. The carbonyl of I-7 is then reduced usingmethods known in the art (e.g., NaBH₄) followed by coupling with acidI-8 (e.g., with DMAP, EDC.Cl) to yield the desired product I-9.

General Reaction Scheme 10 (“Method J”) provides another alternativemethod for preparation of exemplary compounds of Formula (IV) or (V)(i.e., compound “J-6”), wherein R, R¹, a¹, a² and Z are as definedherein. Compounds of structure J-1 are purchased or prepared accordingto methods known in the art. Compound J-1 is used to form J-2 underappropriate conditions (e.g., diethyl acetone dicarboxylate, EtONa).Alcohol J-3 is then coupled to J-2 using standard conditions (e.g.,DMAP, EDC.HCl) to yield J-4. The carbonyl of J-4 is reduced (e.g., withNaBH₄) followed by coupling with acid J-5 (e.g., with DMAP, EDC.HCl) toyield the desired product J-6.

It should be noted that various alternative strategies for preparationof compounds of Formula (IV) and (V) are available to those of ordinaryskill in the art. For example, other compounds of Formula (IV) and (V)wherein G¹ and G² (as disclosed herein) are other than ester can beprepared according to analogous methods using the appropriate startingmaterial. Further, the General Reaction Schemes above depict preparationof a compound of Formula (IV) and (V), wherein R¹ and R² as well as a¹and a² are the same; however, this is not a required aspect of theinvention and modifications to the above reaction scheme are possible toyield compounds wherein R¹ and R² as well as a¹ and a² are different(i.e., resulting in an asymmetric compound). The use of protectinggroups as needed and other modification to the above General ReactionSchemes will be readily apparent to one of ordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

EXAMPLES Example 1 Preparation of Lipid Nanoparticle Compositions

Lipid nanoparticles, cationic lipids and polymer conjugated lipids(PEG-lipid) were prepared and tested according to the general proceduresdescribed in PCT Pub. Nos. WO 2015/199952, WO 2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein byreference, or were prepared as described herein.

Cationic lipid(s), DSPC, cholesterol and PEG-lipid were solubilized inethanol at a desired molar ratio (e.g., 50:0 to 10:28.5 to 38.5:1.5 to1.7 or 47.5:10:40.8:1.7). Lipid nanoparticles (LNP) were prepared at atotal lipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNAwas diluted to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringepumps were used to mix the lipid solution (i.e., LNP componentssolubilized in ethanol) with the mRNA aqueous solution at a ratio ofabout 1:5 to 1:3 (vol/vol) with total flow rates above 15 mL/min. Theethanol was then removed and the external buffer replaced with PBS bydialysis. Finally, the lipid nanoparticles were filtered through a 0.2μm pore sterile filter. Lipid nanoparticle particle size was 55-95 nmdiameter, and in some instances approximately 70-90 nm as determined byquasi-elastic light scattering using a Malvern Zetasizer Nano ZS(Malvern, UK).

Example 2 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Luciferase mRNA in vivo evaluation studies were performed in 6-8 weekold female C57BL/6 mice (Charles River) 8-10 week old CD-1 (Harlan) mice(Charles River) according to guidelines established by an institutionalanimal care committee (ACC) and the Canadian Council on Animal Care(CCAC). Varying doses of mRNA-lipid nanoparticle were systemicallyadministered by tail vein injection and animals euthanized at a specifictime point (e.g., 4 hours) post-administration. Liver and spleen werecollected in pre-weighed tubes, weights determined, immediately snapfrozen in liquid nitrogen and stored at −80° C. until processing foranalysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total μgprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega). For arepresentative formulation having a combination of two cationic lipids,a four-hour time point was chosen for an efficacy evaluation of thelipid formulation.

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express aluciferase protein, originally isolated from the firefly, photinuspyralis. FLuc is commonly used in mammalian cell culture to measure bothgene expression and cell viability. It emits bioluminescence in thepresence of the substrate, luciferin. This capped and polyadenylatedmRNA is fully substituted with 5-methylcytidine and pseudouridine.

Example 3 Activity of Lipid Formulations Compared at Different ComponentRatios

For a given set of lipid components, the interdependent effects on size,polydispersity, activity and encapsulation were determined bysimultaneously varying all four components in controlled manner based onthe principles of statistical design of experiments. This experimentaldesign was generated and the resulting data was analyzed withDesignExpert 9 (StatEase Inc, MN) using the design ranges andconstraints produced by I-Optimal. The mixture experimental design wasbuilt to resolve up to quadratic interaction terms. The designconstraints used compound I-6 as the representative cationic lipid(ranging from 30 to 70 mol %), DSPC (ranging from 5 to 30 mol %),cholesterol (ranging from 5 to 50 mol %), PEG lipid (ranging from 0.5 to3 mol %). When all parameters combine, the total is 100 mol %.

LNP's were formulated by an in-line mixing process as described inExample 1. These formulations were prepared such that the mRNA tocationic lipid ratio was held constant (N/P=6.3). The size andpolydispersity index data were generated using Malvern Nanosizer ZS. Thediameters given are intensity weighted means. Encapsulation wasdetermined using a fluorescent intercalating dye based assay(Ribogreen). Activity data was generated using an in vivo murine modelof mRNA expression based on photinus pyralis (i.e., firefly) luciferaseas described in Example 2.

Table 5 provides results of various design of experiment trials. Thesize and encapsulation results for these LNP's demonstrate that theparticles are generally physically equivalent to each other for themajority of the experimental design space. It is apparent that very highcationic lipid formulations suffer from poor encapsulation and sizecharacteristics regardless of the proportions of the other lipidcomponents.

TABLE 5 Formulation proportions and resultant particle diameter,polydispersity, and encapsulation percentage. Lipid PEG Mean Trial (I-6)DSPC Chol (IVa) diameter Encap. No. % % % % nm PDI % 1 51.1 18.6 28.61.8 72.2 0.078 84.6 2 30.0 30.0 37.0 3.0 54.6 0.133 87.9 3 30.0 18.350.0 1.8 57.5 0.104 93.5 4 63.3 30.0 5.0 1.8 80.5 0.087 48.6 5 51.1 18.628.6 1.8 75.0 0.029 91.3 6 47.3 9.0 40.7 3.0 63.0 0.035 86.3 7 63.3 30.05.0 1.8 78.5 0.093 44.4 8 30.0 18.3 50.0 1.8 56.0 0.097 96.4 9 70.0 16.310.7 3.0 71.8 0.095 17.1 10 51.1 18.6 28.6 1.8 75.4 0.028 83.8 11 40.517.8 39.3 2.4 55.8 0.098 83.3 12 70.0 5.0 22.8 2.2 108.7 0.042 20.6 1341.5 30.0 28.0 0.5 108.2 0.049 97.1 14 42.0 5.0 50.0 3.0 49.3 0.074 91.515 50.0 10.0 38.5 1.5 83.2 0.042 89.6 16 41.5 30.0 28.0 0.5 103.0 0.06091.7 17 70.0 18.0 11.5 0.5 173.3 0.247 39.4 18 70.0 5.0 22.8 2.2 114.00.051 12.7 19 60.5 16.0 21.0 2.4 75.1 0.063 52.8 20 44.5 5.0 50.0 0.5123.9 0.035 95.2 21 51.3 30.0 15.7 3.0 56.3 0.131 49.1

The data from the design of experiments was used to design furtherexperiments to determine the optimal percentage of the various LNPcomponents across a relevant range, i.e., that allows for equivalentdesirable physical characteristics. Table 6 provides size,polydispersity, encapsulation % and activity for various LNPformulations comprising cationic lipid II-10. Table 7 provides size,polydispersity, encapsulation % and activity for various LNPformulations comprising cationic lipid II-9. Table 8 provides size,polydispersity, encapsulation % and activity for various LNPformulations comprising cationic lipid I-5. The activity data in Table 8is reported as relative to the formulations comprising 50 mol % cationiclipid to address variability in activity for different batches ofnominally the same mRNA. The data collectively show that LNPs comprisingbetween 40 and 50 mol % of cationic lipid surprisingly have betteractivity relative to LNPs with cationic lipid outside this range eventhough polydispersity and/or encapsulation are generally equivalentacross the given range.

TABLE 6 Cationic Lipid Titration for Compound II-10 Cationic lipid/ MeanDSPC/ Activity at Chol/ size Encapsulation 1 mg/kg PEGA (nm) PDI (%)(ng/g) 42.5/10/46/1.5 57 0.087 96 8602 45/10/43.5/1.5 61 0.029 97 1197547.5/10/41/1.5 64 0.042 98 15869 50/10/38.5/1.5 71 0.043 97 12613

TABLE 7 Cationic Lipid Titration for Compound II-9 Cationic lipid/ MeanDSPC/ Activity at Chol/ size Encapsulation 0.3 mg/kg PEGA (nm) PDI (%)(ng/g) 42.5/10/46/1.5 60 0.070 95% 2955 45/10/43.5/1.5 62 0.023 97% 268647.5/10/41/1.5 64 0.002 97% 3424 50/10/38.5/1.5 70 0.041 97% 299755/10/33.5/1.5 78 0.002 95% 1557

TABLE 8 Cationic Lipid Titration for Compound I-5 Cat lipid/ DSPC/ MeanChol/ size Activity at Relative to PEGA (nm) PDI Encaps 1 mg/kgbenchmark 42.5/10/46/1.5 71 0.002 96% 4760 0.74 (mRNA batch A)45/10/43.5/1.5 71 0.019 98% 6984 1.08 (mRNA batch A) 47.5/10/41/1.5 680.042 97% 7057 1.10 (mRNA batch A) 50/10/38.5/1.5 71 0.040 97% 6444 1(mRNA batch A) 50/10/38.5/1.5 77 0.033 93% 15260 1 (mRNA batch B)55/9/34.5/1.5 84 0.007 91% 13555 0.89 (mRNA batch B) 60/7.9/30.6/1.5 900.015 90% 12063 0.79 (mRNA batch B) 65/6.9/26.6/1.5 102 0.013 85% 46400.30 (mRNA batch B)

Example 4 Variable Formulation Component Proportions and Results

Similar to Example 3, the interdependent effects of all lipid componentson activity as well as size and encapsulation can be determined bysimultaneously varying all four components in controlled manner based onthe principles of statistical design of experiments. The followingexperimental design was generated and the resulting data was analyzedwith DesignExpert 9 (StatEase Inc, MN) using the design ranges andconstraints given below. The I-Optimal mixture experimental design wasbuilt to resolve up to quadratic interaction terms.

The design constraints used compound II-5 as the cationic lipid (rangingfrom 36 to 59 mol %), DSPC (ranging from 5 to 15 mol %), chloresterol(ranging from 35 to 48 mol %), PEG lipid (ranging from 1 to 2 mol %).When all parameters combine, the total is 100 mol %.

LNP's were formulated by an in-line mixing process as described inExample 1. These formulations were prepared such that the mRNA tocationic lipid ratio was held constant (N/P=6.3). The size andpolydispersity index data were generated using Malvern Nanosizer ZS. Thediameters given are intensity weighted means. Encapsulation wasdetermined using a fluorescent intercalating dye based assay(Ribogreen). Activity data was generated using an in vivo murine modelof mRNA expression based on photinus pyralis (i.e., firefly) luciferaseas described in Example 1.

The data in Table 9 again show that LNPs comprising between 40 and 50mol % of cationic lipid have better activity, polydispersity and/orencapsulation relative to LNPs with cationic lipid outside this range,regardless of variations in the other LNP components within a reasonablerange.

TABLE 9 Formulation Proportions and Experimental Results Cationic MeanLipid PEG Activ- Diam- En- Trial (I-5) Chol DSPC (IVa) ity eter caps.No. % % % % ng/g nm PDI % 1 47.5 41 10 1.5 4103 73.9 0.047 96.5 2 53 3510 2 3243 74.8 0.052 92.7 3 53 41 5 1 3813 85.4 0.054 95.4 4 46.5 47 51.5 2095 63.1 0.040 97.5 5 47.5 41 10 1.5 3742 74.5 0.023 96.1 6 42 4710 1 1911 105.7 0.045 97.9 7 54 35 10 1 2627 98.6 0.026 91.3 8 36 47 152 1982 63.0 0.069 96.7 9 48.5 35 15 1.5 2770 77.1 0.035 94.9 10 52 41 52 3279 71.2 0.050 94.9 11 37 47 15 1 636 110.4 0.054 97.9 12 47.5 41 101.5 3132 72.9 0.036 97.3 13 58.5 35 5 1.5 2741 96.8 0.020 91.3 14 47.2544 7.5 1.25 4385 74.1 0.008 97.5 15 42 41 15 2 3230 65.4 0.044 95.9

Example 5 Synthesis of Compound I-1

Compound I-1 was prepared according to method B as follows:

A solution of octan-1,8-diol (9.8 g) in methylene chloride (100 mL) andtetrahydrofuran (60 mL) was treated with 2-ethylhexanoyl chloride (10g). Triethylamine (15 mL) was slowly added and the solution stirred forthree days. The reaction mixture was filtered and the filtrate washedwith brine (2×). The organic fraction was dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The crude product wasfiltered through silica gel (20 g) using methylene chloride, yielding15.8 g of crude product. The resultant oil was dissolved in methylenechloride (100 mL) and treated with pyridinum chlorochromate (13 g) fortwo hours. Diethyl ether (400 mL) as added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil passed down a silica gel (77 g) column using a ethylacetate/hexane (0-6%) gradient. 8-O-(2′-ethylhexanoyloxy)octanal (6.7 g)was recovered as an oil.

A solution of 8-O-(2′-ethylhexanoyloxy)octanal (6.7 g), acetic acid (25drops) and 2-N,N-dimethylaminoethylamine (0.54 g) in methylene chloride(40 mL) was treated with sodium triacetoxyborohydride (1.5 g) overnight.The solution was washed with aqueous sodium hydrogen carbonate, followedby brine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (75 g) column using a methanol/methylene chloride (0-10%) gradient,followed by a second column (20 g), to yield compound I-1 (1 g) as acolorless oil.

Example 6 Synthesis of Compound I-2

Compound I-2 was prepared according to method A as follows:

Under an argon atmosphere, to a round-bottom flask charged with phytol(593 mg, 2 mmol), 6-bromohexanoic acid (780 mg, 4 mmol) and4-(dimethylamino)pyridine (60 mg) in dichloromethane (20 mL) was addeddicyclohexylcarbodiimide (908 mg, 4.4 mmol). The precipitate wasdiscarded by filtration. The filtrate was concentrated and the resultingresidue was purified by column chromatography on silica gel eluted witha gradient mixture (0% to 3%) of ethyl acetate in hexanes. This gave acolorless oil (0.79 g 1.67 mmol, 83%) of(E)-3,7,11,15-tetramethylhexadec-2-enyl 6-bromohexanoate.

A solution of (E)-3,7,11,15-tetramethylhexadec-2-enyl 6-bromohexanoate(0.42 g, 0.887 mmol), N,N-diisopropylethylamine (1.5 mol eq., 1.33 mmol,MW 129.25, 171 mg) and N,N-dimethylethylenediamine (39 mg, 0.44 mmol) inDMF (4 mL) was heated at 77 C for 18 h. The reaction mixture was thencooled and extracted with hexanes (3×20 mL). The hexane extracts werecombined, dried over sodium sulfate, filtered and concentrated. This iscombined with 2^(nd) reaction (total about 0.7 g). The crude waspurified several times by column chromatography on silica gel elutedwith a gradient mixture (0% to 5%) of methanol in DCM. This gave aslightly yellow oil (39 mg) of the desired product. ¹H NMR (400 MHz,CDCl₃) δ: 5.33 (m, 2H), 4.59 (m, 4H), 2.85-2.25 (m, 18H).

Example 7 Synthesis of Compound I-3

Compound I-3 was prepared in a manner analogous to compound I-2 startingfrom bromoacetic acid, rather than 6-bromohexanoic acid, to yield 22 mgof thick colorless oil, 0.029 mmol, 6%. ¹H NMR (400 MHz, CDCl₃) δ: 5.32(m, 2H), 4.62 (m, 4H), 3.62 (s, 2H), 3.60 (s, 2H), 3.28-2.33 (m, 10H),2.09-2.00 (m, 4H), 1.76 (s, 3H), 1.70 (s, 3H), 1.60-1.47 (m, 6H),1.47-0.97 (32H), 0.89-0.84 (m, 24H).

Example 8 Synthesis of Compound I-4

Compound I-4 was prepared according to method B as follows:

A solution of dodecan-1,12-diol (10 g) in methylene chloride (100 mL)and tetrahydrofuran (50 mL) was treated with 2-ethylhexanoic acid (7.2g), DCC (10.5 g), DMAP (3.5 g) and triethylamine (10 mL). The solutionwas stirred for four days. The reaction mixture was filtered and thefiltrate washed with dilute hydrochloric acid. The organic fraction wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was dissolved in methylene chloride (50 mL),allowed to stand overnight, and filtered. The solvent was removed toyield 12.1 g crude product.

The crude product dissolved in methylene chloride (100 mL) and treatedwith pyridinum chlorochromate (8 g) overnight. Diethyl ether (400 mL) asadded and the supernatant filtered through a silica gel bed. The solventwas removed from the filtrate and resultant oil passed down a silica gel(75 g) column using a ethyl acetate/hexane (0-6%) gradient. Crude12-O-(2′-ethylhexanoyloxy)dodecanal (3.5 g) was recovered as an oil.

A solution of the crude product (3.5 g), acetic acid (60 drops) and2-N,N-dimethylaminoethylamine (0.30 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (0.86 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate, followed bybrine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (20 g) column using a methanol/methylene chloride (0-8%) gradient,followed by a second column (20 g), to yield the desired product as a(0.6 g) as a colorless oil.

Example 9 Synthesis of Compound I-5

Compound I-5 was prepared according to method B as follows:

A solution of hexan-1,6-diol (10 g) in methylene chloride (40 mL) andtetrahydrofuran (20 mL) was treated with 2-hexyldecanoyl chloride (10 g)and triethylamine (10 mL). The solution was stirred for an hour and thesolvent removed. The reaction mixture was suspended in hexane, filteredand the filtrate washed with water. The solvent was removed and theresidue passed down a silica gel (50 g) column using hexane, followed bymethylene chloride, as the eluent, yielding6-(2′hexyldecanoyloxy)hexan-1-ol as an oil (7.4 g).

The purified product (7.4 g) was dissolved in methylene chloride (50 mL)and treated with pyridinum chlorochromate (5.2 g) for two hours. Diethylether (200 mL) as added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oilpassed down a silica gel (50 g) column using a ethyl acetate/hexane(0-5%) gradient. 6-(2′-hexyldecanoyloxy)dodecanal (5.4 g) was recoveredas an oil.

A solution of the product (4.9 g), acetic acid (0.33 g) and2-N,N-dimethylaminoethylamine (0.40 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (2.1 g) for two hours. Thesolution was washed with aqueous sodium hydroxide. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel (50 g) column using amethanol/methylene chloride (0-8%) gradient to yield the desired product(1.4 g) as colorless oil.

Example 10 Synthesis of Compound I-6

Compound I-6 was prepared according to method B as follows:

Compound I-6 was prepared according to method B as follows: A solutionof nonan-1,9-diol (12.6 g) in methylene chloride (80 mL) was treatedwith 2-hexyldecanoic acid (10.0 g), DCC (8.7 g) and DMAP (5.7 g). Thesolution was stirred for two hours. The reaction mixture was filteredand the solvent removed. The residue was dissolved in warmed hexane (250mL) and allowed to crystallize. The solution was filtered and thesolvent removed. The residue was dissolved in methylene chloride andwashed with dilute hydrochloric acid. The organic fraction was driedover anhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was passed down a silica gel column (75 g) using 0-12% ethylacetate/hexane as the eluent, yielding 9-(2′-hexyldecanoyloxy)nonan-1-ol(9.5 g) as an oil.

The product was dissolved in methylene chloride (60 mL) and treated withpyridinum chlorochromate (6.4 g) for two hours. Diethyl ether (200 mL)was added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil passed down asilica gel (75 g) column using a ethyl acetate/hexane (0-12%) gradient,yielding 9-(2′-ethylhexanoyloxy)nonanal (6.1 g) as an oil.

A solution of the crude product (6.1 g), acetic acid (0.34 g) and2-N,N-dimethylaminoethylamine (0.46 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (2.9 g) for two hours. Thesolution was diluted with methylene chloride washed with aqueous sodiumhydroxide, followed by water. The organic phase was dried over anhydrousmagnesium sulfate, filtered and the solvent removed. The residue waspassed down a silica gel (75 g) column using a methanol/methylenechloride (0-8%) gradient, followed by a second column (20 g) using amethylene chloride/acetic acid/methanol gradient. The purified fractionswere dissolved in methylene chloride, washed with dilute aqueous sodiumhydroxide solution, dried over anhydrous magnesium sulfate, filtered andthe solvent removed, to yield the desired product (1.6 g) as colorlessoil.

Example 11 Synthesis of Compound I-7

Compound I-7 was prepared from 3,5,5-trimethylhexyl 10-bromodecanoateand N,N-dimethylethane-1,2-diamine according to method A to yield 144 mgof slightly yellow oil, 0.21 mmol, 11%). ¹H NMR (400 MHz, CDCl₃) δ: 4.09(t-like, 6.6 Hz, 4H), 2.58-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.29(t-like, 7.5 Hz, 4H), 2.25 (s, 6H), 1.67-1.57 (m, 8H), 1.52-1.39 (m,6H), 1.36-1.21 (m, 24H), 0.95 (d, 6.6 Hz, 6H), 0.90 (s, 18H).

Example 12 Synthesis of Compound I-8

Compound I-8 was prepared by method A in 15% yield. ¹H NMR (400 MHz,CDCl₃) δ: 5.11-5.04 (m, 2H), 2.60-2.54 (m, 2H), 2.47-2.36 (m, 6H), 2.27(t-like, 7.4 Hz, 4H), 2.25 (s, 6H), 1.66-1.40 (m, 16H), 1.34-1.23 (m,24H), 0.91 (d, 6.5 Hz, 24H).

Example 13 Synthesis of Compound I-9

Compound I-9 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10.0 g) in methylene chloride (100 mL) wastreated with citroneloyl chloride (10.1 g, prepared from citronelic acidand oxalyl chloride) and triethylamine (10 mL), and stirred for threedays. The reaction mixture was diluted with methylene chloride andwashed with dilute hydrochloric acid. The organic fraction was driedover anhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was taken up in hexane, filtered and the solvent removed. Theresidue was passed down a series of silica gel columns (60-70 g) usinghexane followed by methylene chloride as the eluent, yielding9-(citroneloyloxy)nonan-1-ol (7.6 g) as an oil.

The product was dissolved in methylene chloride (50 mL) and treated withpyridinum chlorochromate (6.4 g) for 90 minutes. Diethyl ether (200 mL)as added and the supernatant filtered through a silica gel bed. Theresidue was dissolved in hexane and passed through a silica gel (20 g)column using hexane as the eluent, yielding 9-(citroneloyloxy)nonanal (5g) as an oil.

A solution of the crude product (5 g), acetic acid (0.33 g) and2-N,N-dimethylaminoethylamine (0.48 g) in methylene chloride (40 mL) wastreated with sodium triacetoxyborohydride (1.2 g) overnight. Thesolution was diluted with methylene chloride and washed with aqueoussodium hydroxide. The organic phase was dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The residue was passed down asilica gel (50 g) column using a 0-12% methanol/methylene chloridegradient, followed by a second silica gel column (20 g) using the samegradient, to yield the desired product (0.6 g) as colorless oil.

Example 14 Synthesis of Compound I-10

Compound I-10 was prepared according to method A to yield 147 mg ofcolorless oil, 0.23 mmol, 17%. ¹H NMR (400 MHz, CDCl₃) δ: 4.11 (t, 6.9Hz, 4H), 2.56-2.52 (m, 2H), 2.44-2.35 (m, 6H), 2.29 (t-like, 7.5 Hz,4H), 2.24 (s, 6H), 1.75-1.66 (m, 8H), 1.66-1.57 (m, 4H), 1.52 (q-like,6.9 Hz, 4H), 1.46-1.38 (m, 4H), 1.38-1.13 (m, 30H), 0.98-0.87 (m, 4H).

Example 15 Synthesis of Compound I-11

Compound I-11 was prepared according to method A to yield 154 mg ofslightly yellow oil, 0.22 mmol, 14%). ¹H NMR (400 MHz, CDCl₃) δ: 4.88(quintet, 6.2 Hz, 2H), 3.20-2.40 (m, 8H), 2.39 (s, 6H), 2.29 (t, 7.5 Hz,4H), 1.67-1.56 (m, 4H), 1.56-1.48 (m, 8H), 1.38-1.21 (m, 44H), 0.92-0.86(m, 12H).

Example 16 Synthesis of Compound I-12

Compound I-12 was prepared according to method A to yield 169 mg ofslightly yellow oil, 0.26 mmol, 17%). ¹H NMR (400 MHz, CDCl₃) δ:4.03-3.95 (ABX pattern, 4H), 2.54 (m, 2H), 2.44-2.35 (m, 6H), 2.30(t-like, 7.5 Hz, 4H), 2.25 (s, 6H), 1.66-1.54 (m, 6H), 1.47-1.23 (m,40H), 0.92-0.88 (m, 12H).

Example 17 Synthesis of Compound I-13

Compound I-13 was prepared according to method A to yield 152 mg ofwhite paste, 0.23 mmol, 16%. ¹H NMR (400 MHz, CDCl₃) δ: 4.03 (t, 6.7 Hz,4H), 3.10-2.41 (very broad, 8H), 2.34 (s, 6H), 2.30 (t, 7.5 Hz, 4H),1.66-1.46 (m, 12H), 1.39-1.21 (m, 40H), 0.89 (t-like, 6.9 Hz, 6H).

Example 18 Synthesis of Compound I-14

Compound I-14 was prepared according to method A to yield 111 mg ofcolorless oil, 0.16 mmol, 11%. ¹H NMR (400 MHz, CDCl₃) δ: 5.09 (m, 2H),4.16-4.05 (m, 4H), 3.10-2.40 (very broad, 8H), 2.31 (s, 6H), 2.29 (t,7.5 Hz, 4H), 2.06-1.89 (m, 4H), 1.69 (d, 0.8 Hz, 6H), 1.61 (s, 6H),1.73-1.13 (m, 50H), 0.92 (d, 6.6 Hz, 6H).

Example 19 Synthesis of Compound I-15

Compound I-15 was prepared according to method A to yield 116 mg ofwhite paste, 0.16 mmol, 10%. ¹H NMR (400 MHz, CDCl₃) δ: 4.06 (t, 6.7 Hz,4H), 2.62-2.51 (broad, 2H), 2.48-2.33 (br., 6H), 2.29 (t, 7.5 Hz, 4H),2.25 (s, 6H), 1.69 (quintet, 7.0 Hz, 8H), 1.48-1.38 (br., 4H), 1.38-1.21(m, 52H), 0.89 (t-like, 6.8 Hz, 6H).

Example 20 Synthesis of Compound I-16

Compound I-16 was prepared according to method A to yield 118 mg ofcolorless oil 0.17 mmol, 12%. ¹H NMR (400 MHz, CDCl₃) δ: 4.06 (t, 6.8Hz, 4H), 2.57-2.52 (m, 2H), 2.44-2.34 (m, 6H), 2.29 (t, 7.6 Hz, 4H),2.25 (s, 6H), 1.62 (quintet-like, 7.0 Hz, 8H), 1.47-1.39 (m, 4H),1.37-1.22 (m, 44H), 0.89 (t-like, 6.8 Hz, 6H).

Example 21 Synthesis of Compound I-17

Compound I-17 was prepared according to method A to yield 145 mg ofslightly yellow oil, 0.21 mmol, 13%. ¹H NMR (400 MHz, CDCl₃) δ: 5.01 (m,0.27H from cis-isomer), 4.63 (tt, 11.2 Hz, 4.5 Hz, 1.73H fromtrans-isomer), 2.61-2.24 (18H), 2.01 (m, 4H), 1.81 (m, 4H), 1.61(quintet-like, 7.2 Hz, 4H), 1.44 (m, 4H), 1.36-1.21 (24H), 1.11 (m, 4H),1.01 (m, 2H), 0.87 (s, 2.7H from cis-isomer), 0.86 (s, 15.3H fromtrans-isomer).

Example 22 Synthesis of Compound I-18

Compound I-18 was prepared according to method A to yield 111 mg ofcolorless oil, 0.17 mmol, 14%. ¹H NMR (400 MHz, CDCl₃) δ: 4.88 (quintet,6.2 Hz, 2H), 2.61-2.51 (br., 2H), 2.48-2.34 (br, 6H), 2.29 (t, 7.6 Hz,4H), 2.25 (s, 6H), 1.62 (quintet-like, 7.3 Hz, 4H), 1.55-1.48 (m, 8H),1.47-1.39 (m, 4H), 1.37-1.21 (m, 32H), 0.91-0.86 (m, 12H).

Example 23 Synthesis of Compound I-19

Compound I-19 was prepared according to method A to yield 76 mg ofcolorless oil, 0.11 mmol, 6%. ¹H NMR (400 MHz, CDCl₃) δ: 5.77 (dt-like,14.4 Hz, 6.6 Hz, 2H), 5.55 (dtt-like, 14.4 Hz, 6.5 Hz, 1.4 Hz, 2H), 4.51(dd, 6.6 Hz, 0.6 Hz, 4H), 2.61-2.50 (br., 2H), 2.50-2.34 (br. 6H), 2.30(t, 7.5 Hz, 4H), 2.25 (s, 6H), 2.04 (q, 7.1 Hz, 4H), 1.62 (quintet, 7.3Hz, 4H), 1.48-1.21 (40H), 0.88 (t-like, 6.8 Hz, 6H).

Example 24 Synthesis of Compound I-20

Compound I-20 was prepared according to the general procedure A to yield157 mg of colorless oil, 0.22 mmol, 14%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.33 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.63 (quintet-like, 7.3 Hz, 6H), 1.43 (quintet-like,7.3 Hz, 4H), 1.36-1.21 (44H), 0.93-0.86 (m, 12H).

Example 25 Synthesis of Compound I-21

Compound I-21 was prepared according to the general procedure A to yield164 mg of colorless oil, 0.21 mmol, 14%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.62 (quintet-like, 7.3 Hz, 6H), 1.43 (quintet-like,7.3 Hz, 4H), 1.36-1.21 (52H), 0.93-0.86 (m, 12H).

Example 26 Synthesis of Compound I-22

Compound I-22 was prepared according to method A as follows:

Step 1

To a solution of 6-bromohexanoic acid (20 mmol, 3.901 g),2-hexyl-1-decanol (1.8 eq, 36 mmol, 8.72 g) and 4-dimethylaminopyridine(DMAP 0.5 eq, 10 mmol, 1.22 g) in DCM (80 mL) was added DCC (1.1 eq, 22mmol, 4.54 g). The resulting mixture was stirred at room temperature for16 hours. The precipitate was discarded by filtration. The filtrate wasconcentrated. The residue was purified by column chromatography onsilica gel eluted with a gradient mixture of ethyl acetate in hexanes (0to 2%). This gave the desired product as a colorless oil (7.88 g, 18.8mmol, 94%)

Step 2

A mixture of the bromide from step 1 (1.34 equiv., 7.88 g, 18.8 mmol),N,N-diisopropylethylamine (1.96 eq, 27.48 mmol, 4.78 mL) andN,N-dimethylethylenediamine (1 eq, 14.02 mmol, 1.236 g, 1.531 mL) inacetonitrile (70 mL) in 250 mL flask equipped with a condenser washeated at 79° C. (oil bath) for 16 hours. The reaction mixture wascooled to room temperature and concentrated. The residue was taken in amixture of ethyl acetate and hexanes (1:9) and water. The phases wereseparated, washed with water (100 mL) and brine. Dried over sodiumsulfate and concentrated (8.7 g oil). The crude (8.7 g oil) was purifiedby column chromatography on silica gel (0 to 3% MeOH in chloroform). Thefractions containing the desired product were combined and concentrated.The residue was dissolved in 1 mL of hexane and filtered through a layerof silica gel (3-4 mm, washed with 8 mL of hexane). The filtrate wasblown to dry with a stream of argon and dried well in vacuo overnight(1.30 g, mmol, %, colorless oil, desired product). ¹H NMR (400 MHz,CDCl₃) δ: 3.96 (d, 5.8 Hz, 4H), 2.55-2.50 (m, 2H), 2.43-2.39 (m, 4H),3.37-3.32 (m, 2H), 2.30 (t, 7.5 Hz, 4H), 2.23 (s, 6H), 1.63(quintet-like, 7.6 Hz, 6H), 1.48-1.40 (m, 4H), 1.34-1.20 (52H), 0.88(t-like, 6.8 Hz, 12H).

Example 27 Synthesis of Compound I-23

Compound I-23 was prepared according to the general procedure A to yield200 mg of colorless oil, 0.24 mmol, 16%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.51 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.67-1.58 (m, 6H), 1.43 (quintet-like, 7.3 Hz, 4H),1.36-1.21 (60H), 0.89 (t-like, 6.8 Hz, 12H).

Example 28 Synthesis of Compound I-24

Compound I-24 was prepared according to the general procedure A to yield138 mg of colorless oil, 0.18 mmol, 12%. ¹H NMR (400 MHz, CDCl₃) δ: 4.90(sixlet-liked, 6.3 Hz, 2H), 2.63-2.33 (br. 8H), 2.27 (t, 7.5 Hz, 4H),2.26 (s, 6H), 1.66-1.57 (m, 4H), 1.51-1.39 (m, 6H), 1.35-1.21 (54H),1.20 (d, 6.2 Hz, 6H), 0.89 (t-like, 6.8 Hz, 6H).

Example 29 Synthesis of Compound I-25

Compound I-25 was prepared according to the general procedure A to yield214 mg of colorless oil, 0.24 mmol, 17%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.8 Hz, 4H), 2.58-2.52 (m, 2H), 2.45-2.35 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.25 (s, 6H), 1.62 (quintet-like, 7.0 Hz, 6H), 1.43 (quintet-like,7.0 Hz, 4H), 1.36-1.21 (68H), 0.89 (t-like, 6.7 Hz, 12H).

Example 30 Synthesis of Compound I-26

Compound I-26 was prepared according to the general procedure A to yield170 mg of colorless oil, 0.21 mmol, 13%. ¹H NMR (400 MHz, CDCl₃) δ:5.42-5.29 (m, 8H), 4.05 (t, 6.8 Hz, 4H), 2.77 (t, 6.5 Hz, 4H), 2.55-2.50(m, 2H), 2.43-2.39 (m, 4H), 2.37-2.32 (m, 2H), 2.29 (t, 7.6 Hz, 4H),2.23 (s, 6H), 2.05 (q, 6.8 Hz, 8H), 1.63 (quintet-like, 7.5 Hz, 8H),1.48-1.40 (m, 4H), 1.39-1.23 (36H), 0.90 (t-like, 6.8 Hz, 6H).

Example 31 Synthesis of Compound I-27

Compound I-27 was prepared according to the general procedure A to yield255 mg of colorless oil, 0.29 mmol, 18%. ¹H NMR (400 MHz, CDCl₃) δ: 3.96(d, 5.8 Hz, 4H), 2.55-2.50 (m, 2H), 2.43-2.39 (m, 4H), 3.37-3.32 (m,2H), 2.30 (t, 7.5 Hz, 4H), 2.23 (s, 6H), 1.63 (quintet-like, 7.6 Hz,6H), 1.48-1.40 (m, 4H), 1.34-1.20 (68H), 0.88 (t-like, 6.8 Hz, 12H).

Example 32 Synthesis of Compound I-28

Compound I-28 was prepared according to the general procedure A to yield248 mg of colorless oil, 0.27 mmol, 19%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.8 Hz, 4H), 2.57-2.52 (m, 2H), 2.44-2.34 (m, 6H), 2.30 (t, 7.5 Hz,4H), 2.24 (s, 6H), 1.67-1.58 (m, 6H), 1.43 (quintet-like, 7.3 Hz, 4H),1.36-1.21 (76H), 0.89 (t-like, 6.8 Hz, 12H).

Example 33 Synthesis of Compound I-29

Compound I-29 was prepared according to the general procedure A to yield181 mg of colorless oil, 0.23 mmol, 17%. ¹H NMR (400 MHz, CDCl₃) δ: 4.87(quintet, 6.3 Hz, 4H), 2.56-2.51 (m, 2H), 2.43-2.34 (m, 6H), 2.27 (t,7.5 Hz, 4H), 2.24 (s, 6H), 1.61 (quintet-like, 7.3 Hz, 4H), 1.55-1.46(m, 8H), 1.46-1.37 (m, 4H), 1.36-1.08 (52H), 0.88 (t-like, 6.8 Hz, 12H).

Example 34 Synthesis of Compound I-30

Compound I-30 was prepared according to the general procedure A to yield88 mg of colorless oil, 0.11 mmol, 3%. ¹H NMR (400 MHz, CDCl₃) δ: 3.97(d, 5.5 Hz, 4H), 2.58-2.51 (m, 2H), 2.49-2.44 (m, 4H), 2.38-2.30 (m,6H), 2.24 (s, 6H), 1.75 (quintet-like, 7.3 Hz, 4H), 1.66-1.54 (m, 2H),1.35-1.06 (64H), 0.89 (t-like, 6.4 Hz, 12H).

Example 35 Synthesis of Compound I-31

Compound I-31 was prepared according to the general procedure C to yield275 mg of slightly yellow oil, 0.30 mmol, total yield 35% for threesteps. ¹H NMR (400 MHz, CDCl₃) δ: 3.97 (d, 5.8 Hz, 4H), 2.63-2.47 (m,8H), 2.45-2.41 (m, 4H), 2.31 (t, 7.5 Hz, 4H), 1.82-1.74 (m, 4H), 1.64(quintet-like, 7.6 Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.18(68H), 0.89 (t-like, 6.8 Hz, 12H).

Example 36 Synthesis of Compound I-32

Compound I-32 was prepared according to method C as follows:

Step 1

To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL, MW 61.08, d1.012) in 15 mL of anhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 eq,1.52 g, 3.62 mmol), potassium carbonate (1.9 eq, 3.62 mmol, 500 mg),cesium carbonate (0.3 eq, 0.57 mmol, 186 mg,) and sodium iodide (10 mg)were added and was heated to reflux for 6 days under argon atmosphere.The solvent was evaporated under reduced pressure and the residue wastaken up in hexanes and washed with water and brine. The organic layerwas separated, dried over anhydrous sodium sulfate, filtered andevaporated under reduced to obtain a colorless oil. The crude productwas purified by flash column chromatography on silica gel (230-400 meshsilica gel, MeOH in chloroform, 0 to 4%) to yield 936 mg of colorlessoil (1.27 mmol, 70%).

Step 2

To a magnetically stirred and ice-cooled solution of 936 mg (1.27 mmol)of the product from step 1 in 2 mL of CHCl₃, was added thionyl chloride(2.9 eq, 3.70 mmol, 440 mg, 270 μL,) in 15 mL of chloroform dropwiseunder an Ar atmosphere. After the completion of addition of SOCl₂, theice bath was removed and the reaction mixture was stirred for 16 hoursat room temperature under an argon atmosphere. Removal of CHCl₃, andSOCl₂ under reduced pressure gave a thick yellow oil.

Step 3

The crude from step 2 was dissolved in THF (20 mL). To the THF solutionwas added pyrrolidine (1.6 mL, 1.36 g, 19 mmol). The sealed mixture washeated at 64° C. overnight. The reaction mixture was concentrated (darkbrown oil). The residue was purified by flash dry column chromatographyon silica gel (MeOH in chloroform, 0 to 4%). This gave the desiredproduct as a slightly yellow oil (419 mg, 0.53 mmol, 83%). ¹H NMR (400MHz, CDCl₃) δ: 3.97 (d, 5.8 Hz, 4H), 2.65-2.47 (m, 8H), 2.45-2.41 (m,4H), 2.31 (t, 7.5 Hz, 4H), 1.81-1.74 (m, 4H), 1.64 (quintet-like, 7.6Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.21 (52H), 0.89 (t-like,6.8 Hz, 12H).

Example 37 Synthesis of Compound I-33

Compound I-33 was prepared according to the general procedure C to yield419 mg of slightly yellow oil, 0.54 mmol, total yield 60% for threesteps. ¹H NMR (400 MHz, CDCl₃) δ: 3.97 (d, 5.8 Hz, 4H), 2.57-2.53 (m,2H), 2.46-2.40 (m, 8H), 2.31 (t, 7.5 Hz, 4H), 2.23 (s, 3H), 1.64(quintet-like, 7.6 Hz, 6H), 1.46 (quintet-like, 7.6 Hz, 4H), 1.36-1.20(52H), 1.06 (t, 7.2 Hz, 3H), 0.89 (t-like, 6.8 Hz, 12H).

Example 38 Synthesis of Compound I-34

Compound I-34 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10 g) in methylene chloride (250 mL) wastreated with 2-ethylhexanoic acid (4.5 g), DCC (7.7 g) and DMAP (4.2 g).The solution was stirred for three days. The reaction mixture wasfiltered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. The residue was suspended in hexane(70 mL) and allowed to settle. The supernatant was decanted and thesolvent removed. The residue was dissolved in hexane, allowed to standat room temperature and then filtered. The solvent was removed and theresidue passed down a silica gel column (50 g) using a 0-10% ethylacetate/hexane gradient, followed by a 0-8% methanol/methylene chloridegradient, yielding 5.6 g of 9-(2′ethylhexanoyloxy)nonan-1-ol as acolorless oil.

The product dissolved in methylene chloride (70 mL) and treated withpyridinium chlorochromate (5 g) for two hours. Diethyl ether (250 mL)was added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil dissolved inhexane. The suspension was filtered through a silica gel plug and thesolvent removed, yielding crude 9-(2′ethylhexanoyloxy)nonanal (3.4 g) asan oil.

A solution of the crude product (3.4 g), acetic acid (0.52 g) and2-N,N-dimethylaminoethylamine (0.33 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (1.86 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulfate, filtered and thesolvent removed, yielding compound I-34 as an oil (0.86 g).

Example 39 Synthesis of Compound I-35

Compound I-35 was prepared according to method B as follows:

A solution of dodecan-1,12-diol (18.1 g) in methylene chloride (90 mL)was treated with citronelic acid (7.5 g), DCC (10.0 g) and DMAP (9.5 g).The solution was stirred overnight. The reaction mixture was filteredand the filtrate washed with dilute hydrochloric acid. The organicfraction was dried over anhydrous magnesium sulfate, filtered and thesolvent removed to yield 12.2 g of crude 12-citroneloyloxydodecan-1-ol.

The crude product dissolved in methylene chloride (60 mL) and treatedwith pyridinum chlorochromate (6.8 g) for three hours. Diethyl ether(200 mL) as added and the supernatant filtered through a silica gel bed.The solvent was removed from the filtrate and resultant oil passed downa silica gel (75 g) column using a ethyl acetate/hexane (0-12%)gradient. Crude 12-citroneloyloxydodecanal (6.2 g) was recovered as anoil.

A solution of the crude product (6.2 g), acetic acid (0.44 g) and2-N,N-dimethylaminoethylamine (0.50 g) in methylene chloride (40 mL) wastreated with sodium triacetoxyborohydride (2.9 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate, followed bybrine. The organic phase was dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (75 g) column using an acetic acid/methanol/methylene chloride(2-0%/0-12%/98-88%) gradient. The purified fractions were washed withaqueous sodium hydrogen carbonate, dried over magnesium sulfate,filtered and the solvent removed, yielding compound I-35 (1.68 g) as anoil.

Example 40 Synthesis of Compound I-36

Compound I-36 was prepared according to the general procedure C to yield108 mg of colorless oil (0.14 mmol). ¹H NMR (400 MHz, CDCl₃) δ: 4.87(quintet, 6.3 Hz, 2H), 2.56-2.51 (m, 2H), 2.45-2.40 (m, 4H), 2.38-2.33(m, 2H), 2.29 (t, 7.5 Hz, 4H), 2.24 (s, 6H), 1.64 (quintet-like, 7.7 Hz,4H), 1.55-1.41 (m, 12H), 1.35-1.18 (m, 52H), 0.89 (t-like, 6.8 Hz, 12H).

Example 41 Synthesis of Compound I-37

Compound I-37 was prepared according to the general procedure C to yield330 mg of colorless oil (0.40 mmol, total yield 80% for three steps). ¹HNMR (400 MHz, CDCl₃) δ: 4.87 (quintet, 6.5 Hz, 2H), 2.64-2.47 (m, 8H),2.45-2.40 (m, 4H), 2.29 (t, 7.5 Hz, 4H), 1.81-1.74 (m, 4H), 1.64(quintet-like, 7.6 Hz, 4H), 1.55-1.41 (m, 12H), 1.35-1.18 (m, 50H), 0.89(t-like, 6.8 Hz, 12H).

Example 42

Synthesis of Compound I-38

Compound I-38 was prepared according to method B as follows:

A solution of nonan-1,9-diol (16 g) in methylene chloride (100 mL) wastreated with 2-butyloctanoic acid (10 g), DCC (10.3 g) and DMAP (6.7 g).The solution was stirred for three days. The reaction mixture wasfiltered and hexane (250 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. The residue was suspended in hexaneand allowed to settle. The supernatant was decanted and the solventremoved (repeated twice). The residue was dissolved in hexane, allowedto stand at room temperature and then filtered. The solvent was removedand the residue passed down a silica gel column (18 g) using methylenechloride, yielding crude 9-(2′-butyloctanoyloxy)nonan-1-ol (17.7 g) asan oil.

The crude product was dissolved in methylene chloride (250 mL) andtreated with pyridinium chlorochromate (11.2 g) overnight. Diethyl ether(750 mL) was added and the supernatant filtered through a silica gelbed. The solvent was removed from the filtrate and resultant oildissolved in hexane (150 mL). The suspension was filtered through asilica gel plug and the solvent removed. The crude product was passeddown a silica gel (80 g) column using a 0-6% ethyl acetate/hexanegradient, yielding 9-(2′-butyloctanoyloxy)nonanal (5.3 g) as an oil.

A solution of the product (5.3 g), acetic acid (0.37 g) and2-N,N-dimethylaminoethylamine (0.47 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (3.35 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (60 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulfate, filtered and thesolvent removed, yielding compound I-38 as an oil (2.3 g).

Example 43 Synthesis of Compound I-39

Compound I-39 was prepared according to method B as follows:

A solution of hexan-1,6-diol (12 g) in methylene chloride (250 mL) wastreated with 2-decyltetradecanoic acid (17.5 g), DCC (11.3 g) and DMAP(6.8 g). The solution was stirred for overnight. The reaction mixturewas filtered and hexane added to the filtrate. The mixture was stirredand the precipitates allowed to settle out. The supernatant was decantedand the solvent removed. The residue was passed down a silica gel column(80 g) using hexane followed by 0-1% methanol/methylene chloride,yielding crude 6-(2′-decyltetradecanoyloxy)hexan-1-ol (5.8 g) as an oil.The crude product was dissolved in methylene chloride (70 mL) andtreated with pyridinium chlorochromate (2.9 g) for two hours. Diethylether (250 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed. The crude product was passed down a silicagel (10 g) column using a 0-5% ethyl acetate/hexane gradient, yielding6-(2′-decyltetradecanoyloxy)hexanal (3.2 g) as an oil.

A solution of the product (3.2 g), acetic acid (0.28 g) and2-N,N-dimethylaminoethylamine (0.15 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (0.98 g) overnight. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulfate, filtered and thesolvent removed, yielding compound I-39 as an oil (1.2 g).

Example 44 Synthesis of Compound I-40

Compound I-40 was prepared according to method B as follows:

A solution of nonan-1,9-diol (10.1 g) in methylene chloride (200 mL) wastreated with 2-octyldodecanoic acid (10.0 g), DCC (8.3 g) and DMAP (5.0g). The solution was stirred for overnight. The reaction mixture wasfiltered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. This process was repeated twice. Theresidue was passed down a silica gel column (75 g) using hexane followedby 4-10% methanol/methylene chloride, yielding crude9-(2′-octyldodecanoyloxy)nonan-1-ol (-11 g) as an oil.

The crude product was dissolved in methylene chloride (70 mL) andtreated with pyridinium chlorochromate (8 g) for two hours. Diethylether (400 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed, to yield the crude product,9-(2′-octyldodecanoyloxy)nonanal (8.4 g), as an oil.

A solution of the product (8.4 g), acetic acid (0.84 g) and2-N,N-dimethylaminoethylamine (0.55 g) in methylene chloride (60 mL) wastreated with sodium triacetoxyborohydride (2.9 g) for two hours. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (75 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulfate, filtered and thesolvent removed, yielding compound I-40 as an oil (3.2 g).

Example 45 Synthesis of Compound I-41

Compound I-41 was prepared according to method B as follows:

A solution of nonan-1,9-diol (9.6 g) in methylene chloride (200 mL) wastreated with 2-decyltetradecanoic acid (8.4 g), DCC (8.6 g) and DMAP(5.0 g). The solution was stirred for overnight. The reaction mixturewas filtered and hexane (200 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and the solvent removed. This process was repeated twice. Theresidue was passed down a silica gel column (75 g) using hexane followedby 4-10% methanol/methylene chloride, yielding crude9-(2′-decyltetradecanoyloxy)nonan-1-ol (6.4 g) as an oil.

The crude product was dissolved in methylene chloride (50 mL) andtreated with pyridinium chlorochromate (5.7 g) for two hours. Diethylether (200 mL) was added and the supernatant filtered through a silicagel bed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelplug and the solvent removed, yielding crude9-(2′-decyltetradecanoyloxy)nonanal (5 g) as an oil.

A solution of the product (5 g), acetic acid (0.45 g) and2-N,N-dimethylaminoethylamine (0.32 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (1.6 g) for two hours. Thesolution was washed with aqueous sodium hydroxide solution. The organicphase was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (50 g) columnusing an acetic acid/methanol/methylene chloride (2-0%/0-12%/98-88%)gradient. The purified fractions were washed with aqueous sodiumhydrogen carbonate, dried over magnesium sulfate, filtered and thesolvent removed, yielding compound I-41 as oil (2.2 g).

Example 46 Synthesis of Compound II-1

Compound II-1 was prepared according to method A from compound II-5 toyield 240 mg of colorless oil, 0.32 mmol, 61%). ¹H NMR (400 MHz, CDCl₃)δ: 5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.39-2.25 (m, 7H), 2.22 (s,6H), 2.06 (q, 6.8 Hz, 8H), 1.53 (quintet, 7.3 Hz, 2H), 1.41-1.11 (54H),0.92-0.87 (m, 9H).

Example 47 Synthesis of Compound II-2

Compound II-2 was prepared according to method A as follows:

Compound II-7 (0.84 g, 0.96 mmol) was dissolved in THF (15 mL) and LAH(2 eq. 1.92 mmol, 73 mg, MW37.95) was added in portions at RT. After thereaction mixture was heated at 60° C. overnight, sodium sulfate hydratewas added. The mixture was stirred for 2 hours, filtered through a layerof silica gel. The filtrate was concentrated to give a slightly yellowoil (0.86 g). The crude product was purified by gravity columnchromatography on silica gel (0 to 4% MeOH in chloroform). This gave thedesired product as a colorless oil (420 mg, 0.49 mmol, 51%). ¹H NMR (400MHz, CDCl₃) δ: 5.43-5.30 (m, 12H), 2.78 (t, 6.4 Hz, 6H), 2.40-2.25 (m,7H), 2.22 (s, 6H), 2.06 (q, 6.8 Hz, 12H), 1.53 (quintet, 7.3 Hz, 2H),1.41-1.10 (58H), 0.90 (t, 6.8 Hz, 9H).

Example 48 Synthesis of Compound II-3

Compound II-3 was prepared according to method A from compound II-8 toyield 123 mg of colorless oil, 0.15 mmol, 41%). ¹H NMR (400 MHz, CDCl₃)δ: 5.43-5.30 (m, 8H), 2.78 (t, 6.5 Hz, 4H), 2.35-2.24 (m, 5H), 2.22 (s,6H), 2.15 (d, 5.5 Hz, 2H), 2.06 (q, 6.8 Hz, 8H), 1.52 (quintet, 7.3 Hz,2H), 1.40-1.09 (65H), 0.92-0.87 (m, 12H).

Example 49 Synthesis of Compound II-5

Compound II-5 was prepared according to method A as follows:

Step 1

3-dimethylamine-1-propylamine (6 mmol, 612 mg) and the ketone II-5a(3.16 g, 6 mmol) were mixed in dichloroethane (25 mL) and then treatedwith sodium triacetoxyborohydride (8.49 mmol, 1.8 g) and AcOH (6 mmol,0.36 g, 0.340 mL). The mixture was stirred at room temperature under anargon atmosphere for 2 days. The reaction mixture was quenched by adding1 N NaOH (ca 20 mL), and the product was extracted with a mixture ofhexane and ethyl acetate (ca 5%). The organic extract was washed withwater/brine (1:1), brine, dried over Na₂SO₄ and concentrated to give thedesired product II-5b as yellow oil (3.55 g). The crude product was usedfor the next step without any further purification.

Step 2

A solution of nonanoyl chloride (212 mg, 1.2 mmol) in benzene (10 mL)was added via syringe to a solution of compound II-5b (600 mg, 0.978mmol) and triethylamine (5 mmol, 0.7 mL, 5 eq) and DMAP (20 mg) inbenzene (10 mL) at RT in 10 min. After addition, the mixture was thendiluted with a mixture of hexane and ethyl acetate (ca 5%), washed withwater, washed with brine, dried over sodium sulfate, filtered andconcentrated. The crude product (0.77 g) was purified by gravity columnchromatography on silica gel (230-400 mesh silica gel, 40 g, MeOH inchloroform, 0 to 4%). This gave the desired product 5 as a colorless oil(563 mg, 0.75 mmol, 76%). ¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H),4.56-4.36 (br., 0.3H, due to slow isomerization about amide bond), 3.64(quintet, 7 Hz, 0.7H), 3.12-3.09 (m, 2H), 2.78 (t, 6.4 Hz, 4H),2.33-2.25 (m, 4H), 2.23, 2.22 (two sets of singlet, 6H), 2.06 (q-like,6.8 Hz, 8H), 1.76-1.66 (m, 4H), 1.50-1.40 (m, 4H), 1.40-1.15 (46H), 0.90(t, 6.7 Hz, 6H), 0.88 (t, 6.8 Hz, 3H).

Example 50 Synthesis of Compound II-6

Compound II-6 was prepared according to the general procedure A to yield0.98 g of slightly yellow oil, 1.13 mmol, 58%. ¹H NMR (400 MHz, CDCl₃)δ: 5.43-5.30 (m, 12H), 4.55-4.32 (br., 0.3H, due to slow isomerizationabout amide bond), 3.63 (quintet-like, 7 Hz, 0.7H), 3.15-3.09 (m, 2H),2.78 (t, 6.4 Hz, 6H), 2.33-2.25 (m, 4H), 2.22, 2.23 (two sets ofsinglet, 6H), 2.06 (q-like, 6.8 Hz, 12H), 1.76-1.60 (m, 4H), 1.49-1.16(54H), 0.90 (t-like, 6.8 Hz, 9H).

Example 51 Synthesis of Compound II-7

Compound II-7 was prepared according to method A as follows:

To a solution of 2-ethylheptanoic acid (1.5 eq. 0.83 mmol, 130 mg) inbenzene (6 mL) and DMF (5-10 μL) was added oxalyl chloride (5 eq, 2.8mmol, 349 mg, 0.24 mL) at room temperature. The mixture was stirred atroom temperature for 30 min and then heated at 60° C. for 2 h underargon atmosphere. The mixture was concentrated. The residue was taken upin benzene (6 mL) and concentrated again to remove any oxalyl chloride.The residual oil (light yellow) was taken in 4 mL of benzene and addedvia syringe to a solution of compound II-5b (1 eq., 0.55 mmol, 337 mg)and triethylamine (5 eq, 2.8 mmol, 283 mg, 390 uL) and DMAP (10 mg) inbenzene (6 mL) at room temperature in 10 min. After addition, theresulting mixture was stirred at room temperature overnight. TLC showedthat there was not much reaction. The reaction was concentrated anddried well and used in the following. The residue was taken up in DCM(20 mL). DMAP (200 mg, 1.64 mmol) was added, followed by addition of DCC(1.64 mmol, 338 mg). The mixture was stirred for 11 days and filtered.The filtrate was washed with 5% NaOH (100 mL). The organic phase waswashed with brine, dried over sodium sulfate. Filtration andconcentration gave light brown oil (0.89 g). The crude product (0.89 g)was purified by column chromatography on silica gel (0 to 4% MeOH inchloroform). This gave the desired product as a colorless oil (122 mg,0.16 mmol, 29%). ¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H), 4.69-4.51(very br., estimated 0.4H, due to slow isomerization about amide bond),3.72 (quintet-like, 6.9 Hz, 0.6H), 3.19-3.09 (m, 2H), 2.78 (t, 6.4 Hz,4H), 2.55 (quintet-like, 6.5 Hz, 0.5H), 2.42 (quintet-like, 6.5 Hz,0.5H), 2.29 (q-like, but could be two overlap triplets, 6.9 Hz, 2H),2.24, 2.23 (two sets of singlet, integration ratio is about 1:1, 6H),2.09-2.02 (m, 8H), 1.77-1.58 (m, 4H), 1.55-1.15 (48H), 0.93-0.85 (m,12H).

Example 52 Synthesis of Compound II-8

Compound II-8 was prepared according to the general procedure A to yield0.39 g of colorless oil, 0.46 mmol, 56%. ¹H NMR (400 MHz, CDCl₃) δ:5.43-5.30 (m, 8H), 4.55-4.32 (very br., estimated 0.3H, due to slowisomerization about amide bond), 3.71 (quintet-like, 7 Hz, 0.7H),3.17-3.08 (m, 2H), 2.78 (t, 6.4 Hz, 4H), 2.59 (quintet-like, 6.5 Hz,0.5H), 2.46 (quintet-like, 6.5 Hz, 0.5H), 2.40 (t, 7 Hz, 1H), 2.31 (t, 7Hz, 1H), 2.28, 2.25 (two sets of singlet, integration ratio is about1:1, 6H), 2.09-2.02 (m, 8H), 1.79-1.69 (m, 2H), 1.66-1.57 (m, 2H),1.55-1.16 (62H), 0.92-0.86 (m, 12H).

Example 53 Synthesis of Compound II-9

Compound II-9 was prepared according to method D as follows:

Step 1

3-dimethylamine-1-propylamine (1 eq. 1.3 mmol, 133 mg, 163 μL; MW102.18,d 0.812) and the ketone II-9a (1 eq, 0.885 g, 1.3 mmol) were mixed indichloroethane (8 mL) and then treated with sodium triacetoxyborohydride(1.4 eq, 1.82 mmol, 386 mg; MW 211.94) and AcOH (1 eq., 1.3 mmol, 78 mg,74 μL, MW 60.05, d 1.06). The mixture was stirred at RT under an Aratmosphere for 2 days. The reaction mixture was diluted withhexanes-EtOAc (9:1) and quenched by adding 0.1 N NaOH (20 mL). Theorganic phase was separated, washed with sat NaHCO₃, brine, dried oversodium sulfate, decanted and concentrated to give the desired productII-9b as a slightly yellow cloudy oil (1.07 g, 1.398 mmol).

Step 2

A solution of nonanoyl chloride (1.3 eq, 1.27 mmol, 225 mg) in benzene(10 mL) was added via syringe to a solution of the compound 9b from step1(0.75 g, 0.98 mmol) and triethylamine (5 eq, 4.90 mmol, 0.68 mL) andDMAP (20 mg) in benzene (10 mL) at RT in 10 min. After addition, themixture was stirred at RT overnight. Methanol (5.5 mL) was added toremove excess acyl chloride. After 3 h, the mixture was filtered througha pad of silica gel (1.2 cm). Concentration gave a colorless oil (0.70g). The crude product (0.70 g) was purified by flash dry columnchromatography on silica gel (0 to 4% MeOH in chloroform). This yielded457 mg of colorless oil, 0.50 mmol, 51%. ¹H NMR (400 MHz, CDCl₃) δ:4.54-4.36 (very br., estimated 0.3H, due to slow isomerization aboutamide bond), 3.977, 3.973 (two sets of doublets, 5.8 Hz, 4H), 3.63(quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 10H), 1.49-1.39 (m,4H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 54 Synthesis of Compound II-10

Compound II-10 was prepared according to the general procedure D toyield 245 mg of colorless oil, 0.27 mmol, total yield 53% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.87 (quintet-like, 6.3 Hz, 2H), 4.54-4.36(very br., estimated 0.3H, due to slow isomerization about amide bond),3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m,12H), 1.37-1.11 (60H), 0.92-0.86 (m, 15H).

Example 55 Synthesis of Compound II-11

Compound II-11 was prepared according to the general procedure D toyield 239 mg of colorless oil, 0.26 mmol, total yield 52% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.87 (quintet-like, 6.3 Hz, 2H), 4.54-4.36(very br., estimated 0.3H, due to slow isomerization about amide bond),3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m, 2H), 2.33-2.25 (m, 8H),2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56 (m, 8H), 1.55-1.39 (m,12H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 56 Synthesis of Compound II-12

Compound II-12 was prepared according to the general procedure D toyield 198 mg of colorless oil, 0.20 mmol, total yield 46% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.54-4.36 (very br., estimated 0.3H, due toslow isomerization about amide bond), 3.974, 3.971 (two sets ofdoublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m,2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56(m, 10H), 1.49-1.39 (m, 4H), 1.37-1.11 (76H), 0.92-0.86 (m, 15H).

Example 57 Synthesis of Compound II-13

Compound II-13 was prepared according to the general procedure A toyield 217 mg of colorless oil, 0.21 mmol, total yield 49% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.54-4.36 (very br., estimated 0.3H, due toslow isomerization about amide bond), 3.973, 3.970 (two sets ofdoublets, 5.8 Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.14-3.09 (m,2H), 2.33-2.25 (m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.76-1.56(m, 10H), 1.49-1.39 (m, 4H), 1.37-1.11 (78H), 0.92-0.86 (m, 15H).

Example 58 Synthesis of Compound II-14

Compound II-14 was prepared according to the general procedure A toyield 263 mg of colorless oil, 0.29 mmol, total yield 39% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.54-4.36 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.977, 3.973 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43(m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H),1.49-1.38 (m, 4H), 1.37-1.11 (60H), 0.92-0.86 (m, 15H).

Example 59 Synthesis of Compound II-15

Compound II-15 was prepared according to the general procedure A toyield 234 mg of colorless oil, 0.25 mmol, total yield 34% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.54-4.36 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.977, 3.973 (two sets of doublets, 5.8Hz, 4H), 3.63 (quintet-like, 6.8 Hz, 0.7H), 3.17-3.10 (m, 2H), 2.53-2.43(m, 6H), 2.34-2.26 (m, 6H), 1.83-1.71 (m, 6H), 1.70-1.57 (m, 8H),1.49-1.38 (m, 4H), 1.37-1.11 (62H), 0.92-0.86 (m, 15H).

Example 60 Synthesis of Compound II-16

Compound II-16 was prepared according to method B as follows:

To a solution of the acid 018-19 (0.5 g, 0.90 mmol),N-hydroxysuccinimide (1.2 eq, 1.08 mmol, 124 mg) and DMAP (0.3 eq, 0.27mmol, 33 mg) in DCM (20 mL) was added DCC (2 eq, 1.8 mmol, 371 mg). Theresulting mixture was stirred at room temperature for 16 hours. Thereaction mixture was then filtered and added into a solution of theamine 021-24 (1.26 mmol, 288 mg) in DCM (10 mL) and triethylamine (5mmol, 696 μL). After 15 days, the mixture was concentrated. The residuewas taken up in hexane/ethyl acetate/Et₃N (ca 9:1:0.3) and was filteredthrough a small pad of silica gel, washed with a mixture of hexane/ethylacetate/Et₃N (ca 9:1:0.3). The filtrate was concentrated and a yellowoil was obtained (580 mg). The yellow oil was purified by columnchromatography on silica gel (eluted with a gradient mixture of MeOH inChloroform, 0 to 4.2%). This gave the desired product as a colorless oil(102 mg, 0.13 mmol, 14%). ¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H),3.38-3.29 (m, 3H), 3.28-3.23 (m, 1H), 2.78 (t, 6.4 Hz, 4H), 2.56-2.47(m, 1H), 2.30-2.24 (m, 2H), 2.23, 2.22 (two sets of singlet, 6H),2.09-2.02 (m, 8H), 1.71 (quintet-like, 7.4 Hz, 2H), 1.66-1.48(overlapped with water; estimated 4H), 1.47-1.18 (m, 50H), 0.92-0.86 (m,9H).

Example 61 Synthesis of Compound II-24

Compound II-24 was prepared according to the general procedure A toyield 279 mg of slightly yellow oil, 0.29 mmol, total yield 44% for 2steps. ¹H NMR (400 MHz, CDCl₃) δ: 4.88 (quintet-like, 6.3 Hz, 3H), 3.62(quintet-like, 6.8 Hz, 1H), 3.14-3.08 (m, 2H), 2.33-2.25 (m, 10H), 2.23,2.22 (two sets of singlets, 6H), 1.76-1.58 (m, 10H), 1.52 (q-like, 6.7Hz, 12H), 1.49-1.39 (m, 4H), 1.38-1.14 (50H), 0.89 (t-like, 18H).

Example 62 Synthesis of Compound II-35

Compound II-35 was prepared according to the general procedure A toyield 260 mg of slightly yellow oil, 0.29 mmol, total yield 33% for 2steps. ¹H NMR (400 MHz, CDCl₃) δ: 4.66-4.52 (very br., estimated 0.3H,due to slow isomerization about amide bond), 3.977, 3.973 (two sets ofdoublets, 5.8 Hz, 4H), 3.71 (quintet-like, 6.8 Hz, 0.7H), 3.19-3.09 (m,2H), 2.54, 2.42 (two sets of quintet-like, 6.8 Hz, integration ratio isabout 1:1.2, 1H), 2.33-2.25 (m, 6H), 2.24, 2.22 (two sets of singlet,6H), 1.77-1.11 (74H), 0.93-0.85 (m, 18H).

Example 63 Synthesis of Compound II-17

Compound II-17 was prepared according to method C as follows:

Step 1

3-dimethylamino-1-propylamine (1 eq. 4.14 mmol, 423 mg, 521 μL) andketone II-17a (1 eq., 2.0 g, 4.14 mmol) were mixed in DCE (30 mL) andthen treated with sodium triacetoxyborohydride (1.4 eq., 5.80 mmol,1.229 g) and AcOH (1 eq., 4.14 mmol, 249 mg, 235 The mixture was stirredat room temperature under argon atmosphere for 2 days.

The reaction mixture was diluted with a mixture of hexanes and ethylacetate (9:1, 200 mL) and quenched by adding dilute NaOH solution (0.1N, 270 mL). The two phases were separated. The organic phase was washedwith sat NaHCO₃, brine, dried over sodium sulfate and filtered through apad of silica gel. The pad was washed with 200 mL of a mixture of hexaneand EtOAc (9:1). Then the pad was washed 200 mL of a mixture ofDCM/MeOH/Et3N (85:15:1). The DCM/MeOH/Et3N washing was concentrated togive the desired product (II-17b) as a colorless oil (1.749 g, 3.07mmol, 74%).

Step 2

A solution of nonanoyl chloride (0.333 mL) in benzene (10 mL) was addedto a solution of compound II-17b (0.75 g) and triethylamine (0.92 mL)and DMAP (20 mg) in benzene (20 mL) at room temperature. The mixture wasstirred at room temperature overnight. MeOH (1 mL) was added and themixture continued to stir for 2 hours. The reaction mixture was filteredthrough a pad of silica gel. Concentration of the filtrate gave thedesired product (II-17c) as a yellow oil (0.945 g).

Step 3

To a flask containing 17c (0.945 g, 1.33 mmol and EtOH (25 mL) was addedp-toluenesulfonic acid hydrate (1.33 mmol, 253 mg) at room temperature.The resulting mixture was stirred overnight at RT. The reaction mixturewas heated at 85° C. for 2 hours. More PTSA (160 mg) was added and thereaction mixture continued to heat at 75° C. overnight. The mixture wasconcentrated. The residue was taken up in DCM and washed with diluteNH₄OH solution. The organic phase was washed with a mixture of satsodium bicarbonate and brine; dried over sodium sulfate. Concentrationgave the desired product (II-17d) as a slightly yellow viscous oil(0.799 g, 1.47 mmol). The crude product was purified by silica gelcolumn chromatography (0 to 15% methanol in DCM with trace oftriethlyamine). This gave II-17d as a colorless oil (647 mg, 1.20 mmol,90%).

Step 4

To a solution of II-17d (216 mg, 0.40 mmol), 2-butyloctanoic acid (5 eq,2 mmol, 401 mg), and 4-dimethylaminopyridine (DMAP) (5.5 eq. 2.2 mmol,269 mg) in dichloromethane (20 mL) was added DCC (5.5 eq, 2.2 mmol, 454mg). After being stirred over for 4 days, 3 mL of MeOH was added. Themixture continued to stir for another 16 h. The mixture was filtered andthe filtrate was concentrated to dryness. The crude product was purifiedby gravity column chromatography on silica gel (MeOH in DCM, 0 to 6%).This gave the desired compound II-17 as a slightly yellow oil (colorlessoil, 175 mg, 0.19 mmol, 48%). ¹H NMR (400 MHz, CDCl₃) δ: 4.07, 4.06 (twosets of triplets, 6.7 Hz, 4H), 3.64 (quintet-like, 6.8 Hz, 1H),3.21-3.09 (two sets of multiplets, 2H), 3.00-2.37 (br. 6H), 2.36-2.20(m, 6H), 2.05-1.85 (m, 2H), 1.79-1.53 (m, 10H), 1.52-1.39 (m, 8H),1.37-1.03 (58H), 0.91-0.86 (m, 15H).

Example 64 Synthesis of Compound II-36

Compound II-36 was prepared according to the general procedure C toyield 156 mg of colorless oil, 0.15 mmol, 38% for the last step. ¹H NMR(400 MHz, CDCl₃) δ: 4.07 (triplets, 6.7 Hz, 4H), 3.65 (quintet-like, 6.8Hz, 1H), 3.21 (t-like, 6.8 Hz, 2H), 3.10-3.03 (br. 2H), 2.79, 2.78 (twosets of singlet, 6H), 2.35-2.28 (m, 4H), 2.09 (quintet-like, 7.5 Hz,2H), 1.67-1.54 (m, 10H), 1.54-1.38 (m, 8H), 1.38-1.03 (74H), 0.91-0.86(m, 15H).

Example 65 Synthesis of Compound II-37

Compound II-37 was prepared according to the general procedure A toyield 397 mg of colorless oil, 0.49 mmol, total yield 60% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 5.43-5.30 (m, 8H), 4.13 (q, 7.1 Hz, 2H),4.56-4.34 (br. 0.3H), 3.63 (quintet-like, 6.9 Hz, 0.7H), 3.15-3.08 (m,2H), 2.78 (t-like, 6.4 Hz, 4H), 2.39-2.21 (m, 12H), 2.06 (q-like, 6.9Hz, 8H), 1.79-1.55 (m, 6H), 1.50-1.40 (m, 4H), 1.40-1.15 (m, 45H), 0.90(t-like, 6.8 Hz, 6H).

Example 66 Synthesis of Compound II-38

Compound II-38 was prepared according to method A as follows:

Step 1

To a solution of II-38a (1 eq., 1.266 g, 1.79 mmol) in DCE (15 mL) wasadded 3-dimethylamino-1-propylamine (1 eq. 1.79 mmol, 183 mg, 225 μL),followed by addition of sodium triacetoxyborohydride (1.4 eq., 2.51mmol, 531 mg) and AcOH (1 eq., 1.79 mmol, 107 mg, 101 μL). The mixturewas stirred at room temperature under argon atmosphere for 3 days.

The residue was diluted with hexanes-EtOAc (9:1, 150 mL) and washed withdilute NaOH solution (0.12 N, 100 mL), sat NaHCO₃, brine and dried oversodium sulfate. The organic phase was filtered through a pad of silicagel. The pad was washed with 200 mL of a mixture of hexane and EtOAc(9:1). Then the pad was washed with 200 mL of a mixture of DCM/MeOH/Et₃N(85:15:1). The DCM/MeOH/Et₃N washing was concentrated and dried on highvacuum line to give the desired product (II-38b) as a colorless oil (1.1g, 1.38 mmol, 77%).

Step 2

A solution of nonanoyl chloride (1.5 eq., 0.68 mmol, 120 mg) in benzene(5 mL) was added to a solution of II-38b (0.45 mmol, 360 mg) andtriethylamine (5 eq, 2.25 mmol, 228 mg, 314 μL) and DMAP (10 mg) inbenzene (10 mL) at room temperature in 2 min under argon atmosphere.After addition, the mixture was stirred at room temperature overnight.MeOH (1 mL) was added and the mixture continued to stir 2 h. The crudewas filtered through a pad of silica gel. The filtrate was concentrated.The residue (457 mg) was purified by flash column chromatography onsilica gel (230-400 mesh silica gel, 40 g, MeOH in chloroform, 0 to4.6%). This gave the desired product, compound II-38 as a colorless oil(410 mg, 0.44 mmol, 98%). ¹H NMR (400 MHz, CDCl₃) δ: 4.61-4.35 (br.,estimated 0.4H, due to slow isomerization about amide bond), 3.974,3.964 (two sets of doublets, 5.7 Hz, 4H), 3.64 (quintet-like, 7.0 Hz,0.6H), 3.14-3.08 (m, 2H), 2.34-2.25 (m, 8H), 2.23 (broad s, 6H),1.77-1.58 (m, 10H), 1.53-1.39 (m, 4H), 1.37-1.15 (66H), 0.92-0.86 (m,15H).

Example 67 Synthesis of Compound II-39

Compound II-39 was prepared according to the general procedure A toyield 370 mg of colorless oil, 0.40 mmol, total yield 69% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.61-4.35 (br., estimated 0.4H, due to slowisomerization about amide bond), 3.974, 3.964 (two sets of doublets, 5.7Hz, 4H), 3.64 (quintet-like, 7.0 Hz, 0.6H), 3.14-3.08 (m, 2H), 2.34-2.25(m, 8H), 2.230, 2.221 (two sets of singlet, 6H), 1.75-1.58 (m, 10H),1.51-1.39 (m, 4H), 1.37-1.15 (64H), 0.92-0.86 (m, 15H).

Example 68 Synthesis of Compound II-40

Compound II-40 was prepared according to the general procedure A toyield 382 mg of colorless oil, 0.39 mmol, total yield 68% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.60-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 4.13 (q, 7.2 Hz, 2H), 3.973, 3.964 (twosets of doublets, 5.7 Hz, 4H), 3.63 (quintet-like, 7.0 Hz, 0.7H),3.14-3.08 (m, 2H), 2.34-2.25 (m, 10H), 2.229, 2.220 (two sets ofsinglet, 6H), 1.75-1.58 (m, 12H), 1.51-1.39 (m, 4H), 1.37-1.15 (64H),0.89 (t-like, 7.8 Hz, 12H).

Example 68 Synthesis of Compound II-41

Compound II-41 was prepared according to the general procedure A toyield 309 mg of colorless oil, 0.30 mmol, total yield 73% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.60-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.972, 3.962 (two sets of doublets, 5.7Hz, 4H), 3.64 (quintet-like, 7.1 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.34-2.25(m, 8H), 2.23, 2.22 (two sets of singlet, 6H), 1.75-1.58 (m, 10H),1.51-1.39 (m, 4H), 1.35-1.21 (82H), 0.92-0.86 (m, 15H).

Example 70 Synthesis of Compound II-42

Compound II-42 was prepared according to the general procedure A toyield 235 mg of colorless oil, 0.23 mmol, total yield 56% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.75-4.49 (br., estimated 0.4H, due to slowisomerization about amide bond), 3.97, 3.96 (two sets of doublets, 5.3Hz, 4H), 3.72 (quintet-like, 7 Hz, 0.6H), 3.21-3.05 (m, 2H), 2.53, 2.42(two sets of quintet-like, 6.6 Hz, integration ratio is about 1:1.7,1H), 2.32-2.25 (m, 6H), 2.24, 2.22 (two sets of singlet, 6H), 1.78-1.56(m, 10H), 1.53-1.39 (m, 6H), 1.38-1.17 (76H), 0.93-0.85 (m, 18H).

Example 71 Synthesis of Compound II-43

Compound II-43 was prepared according to the general procedure C toyield 187 mg of colorless oil, 0.23 mmol, 57% for the last step. ¹H NMR(400 MHz, CDCl₃) δ: 4.077, 4.071 (two sets of triplets, 6.7 Hz, 4H),4.56-4.34 (br. 0.3H), 3.64 (quintet-like, 6.9 Hz, 0.7H), 3.15-3.09 (m,2H), 2.34-2.24 (m, 6H), 2.234-2.224 (two sets of singlet, 6H), 1.76-1.58(m, 10H), 1.55-1.39 (m, 8H), 1.39-1.10 (48H), 0.92-0.86 (m, 15H).

Example 72 Synthesis of Compound II-44

Compound II-44 was prepared according to the general procedure A toyield 260 mg of colorless oil, 0.22 mmol, total yield 53% for 2 steps.¹H NMR (400 MHz, CDCl₃) δ: 4.59-4.35 (br., estimated 0.3H, due to slowisomerization about amide bond), 4.03-3.95 (m, 6H), 3.63 (quintet-like,6.9 Hz, 0.7H), 3.14-3.08 (m, 2H), 2.33-2.24 (m, 10H), 2.229, 2.221 (twosets of singlet, 6H),1.75-1.57 (m, 12H), 1.51-1.40 (m, 4H), 1.40-1.08(87H), 0.92-0.86 (m, 18H).

Example 73 Synthesis of 6-(2′-hexyldecanoyloxy)hexan-1-al

A solution of hexan-1,6-diol (27.6 g) in methylene chloride (475 mL) wastreated with 2-hexyldecanoic acid (19.8 g), DCC (18.2 g) and DMAP (11.3g). The solution was stirred for three days. The reaction mixture wasfiltered and hexane (500 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and washed with dilute hydrochloric acid. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved, yielding 30 g of crude product.

The crude product dissolved in methylene chloride (200 mL) and treatedwith pyridinium chlorochromate (15 g) for two hours. Diethyl ether (600mL) was added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil dissolved inhexane. The suspension was filtered through a silica gel plug and thesolvent removed. The residue was passed down a silica gel column (80 g)using hexane, followed by methylene chloride, as the eluent.6-(2′-hexyldecanoyloxy)hexan-1-al (24 g) was obtained as a colorlessoil.

Example 74 Synthesis of 4-(2′-hexyldecanoyloxy)butan-1-al

A solution of butan-1,4-diol (12.5 g) in methylene chloride (200 mL) wastreated with 2-hexyldecanoic acid (9.2 g), DCC (8.8 g) and DMAP (4.9 g).The solution was stirred overnight. The reaction mixture was filteredand the solvent removed. The residue was dissolved in methylene chlorideand washed with dilute hydrochloric acid. The organic phase was driedover anhydrous magnesium sulfate, filtered through a silica gel bed, andthe solvent removed.

The crude product was dissolved in methylene chloride (150 mL) andtreated with pyridinium chlorochromate (6 g) for one hour. Diethyl ether(450 mL) was added and the supernatant filtered through a silica gelbed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelbed and the solvent removed, yielding 4-(2′-hexyldecanoyloxy)butan-1-al(11 g) was obtained as a colorless oil.

Example 75 Synthesis of Compound III-1

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid(0.21 g) and ethanolamine (0.14 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (1.4 g) overnight. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound III-1 as colorless oil (0.63 g).

Example 76 Synthesis of Compound III-2

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid(0.33 g) and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for one hour. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound III-2 as colorless oil (1.1 g).

Example 77 Synthesis of Compound III-3

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.33 g) and 4-aminobutan-1-ol (0.23 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound III-3 as colorless oil (0.4 g).

Example 78 Synthesis of Compound III-4

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.30 g) and 4-aminobutan-1-ol (0.22 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-10/98-90%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound III-4 as colorless oil (0.9 g)

Example 79 Synthesis of Compound III-5

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.31 g) and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.4 g) for one hour. Thesolution was washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-8/98-92%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound III-5 as a colorless oil (0.57 g).

Example 80 Synthesis of Compound III-6

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.30 g) and ethanolamine (0.14 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-10/100-90%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-9/98-92%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound III-6 as colorless oil (0.2 g).

Example 81 Synthesis of Compound III-7

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.14 g) and 5-aminopentan-1-ol (0.24 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound III-7 as colorless oil (0.5 g).

Example 82 Synthesis of Compound III-8

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.17 g) and 6-aminohexan-1-ol (0.26 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound III-8 as colorless oil (0.5 g).

Example 83 Synthesis of Compound III-9

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g) andtrans-2-aminocyclohexanol hydrochloride (0.35 g) in methylene chloride(10 mL)/tetrahydrofuran (10 mL) was treated with sodiumtriacetoxyborohydride (1.3 g) for 1.5 hours. The solution was washedwith aqueous sodium hydrogen carbonate solution. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel column using amethanol/methylene chloride (0-8/100-92%) gradient, yielding compoundIII-9 as colorless oil (0.6 g).

Example 84 Synthesis of Compound III-10

To a solution of 2-aminoethanol (106 mg, 1.75 mmol) in anhydrous THF (15mL), 2-octyldodecyl 6-bromohexanoate (2 eq, 1.66 g, 3.5 mmol), potassiumcarbonate (2 eq, 3.5 mmol, 477 mg,) and cesium carbonate (0.3 eq, 0.525mmol, 171 mg,) were added and was heated at 63° C. (oil bath) for 16 h.Trace of tetrabutylammonium iodide was added to the mixture and themixture was heated to reflux for another 4 days. The solvent wasevaporated under reduced pressure and the residue was taken in a mixtureof hexanes and ethyl acetate (ca 9:1) and washed with water and brine.The organic layer was separated and dried over anhydrous sodium sulfate,filtered and evaporated under reduced to obtain an oil (1.6 g). Theresidue (1.6 g) was purified by column chromatography on silica gel(MeOH in chloroform, 0 to 4%). This gave compound III-10 as colorlessoil (700 mg, 0.82 mmol, 47%).

Example 85 Synthesis of Compound III-11

To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 μL) in 15 mL ofanhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 eq, 1.52 g, 3.62mmol), potassium carbonate (1.9 eq, 3.62 mmol, 500 mg), cesium carbonate(0.3 eq, 0.57 mmol, 186 mg,) and sodium iodide (10 mg) were added andwas heated to reflux for 6 days under argon. The solvent was evaporatedunder reduced pressure and the residue was taken up in hexanes andwashed with water and brine. The organic layer was separated, dried overanhydrous sodium sulfate, filtered and evaporated under reduced pressureto obtain colorless oil. The crude product was purified by flash columnchromatography on silica gel (MeOH in chloroform, 0 to 4%) to yieldcompound III-11 as colorless oil (936 mg, 1.27 mmol, 70%).

Example 86 Synthesis of Compound II-12

Compound III-12 was prepared in a manner analogous to the procedure forCompound III-11 to yield 538 mg of colorless oil, 0.86 mmol, 57%.

Example 87 Synthesis of Compound III-13

To a solution of 2-aminoethanol (171 mg, 2.81 mmol, 169 μL) in anhydrousTHF (30 mL), 2-octyldodecyl 4-bromobutyrate (1.9 eq, 2.386 g, 5.33mmol), potassium carbonate (1.9 eq, 5.33 mmol, 736 mg), cesium carbonate(0.3 eq, 0.84 mmol, 275 mg) and sodium iodide (10 mg) were added and washeated to reflux for 16 h under argon. TLC (Hexane/Ethyl acetate=9:1,CHCl₃/MeOH=19:1) showed that significant amount of 2-octyl-1-dodecanolwas produced. The mixture was cooled and filtered. The filtrate wasconcentrated and the residue was dissolved in 2-octyl-1-dodecanol (2.1g). A few beads of 4 Å molecular sieves and N,N-diisopropylethylamine(1.9 eq, 5.33 mmol, 683 mg, 0.92 mL) was added. The mixture was sealedand heated at 62° C. for another 4 days. The reaction mixture wascooled. Hexane was added. The hexane solution was decanted andconcentrated to dryness. The residue was purified by by columnchromatography on silica gel (MeOH in chloroform, 0 to 4%) to yieldcompound III-13 as colorless oil (282 mg, 0.35 mmol, 13%).

Example 88 Synthesis of Compound III-14

To a solution of heptadecan-9-yl 6-bromohexanoate (2 eq, 1.13 g, 2.61mmol) in anhydrous THF (15 mL), was added 2-aminoethanol (1 eq, 1.31mmol, 79.7 mg), potassium carbonate (2 eq, 2.61 mmol, 361 mg,), cesiumcarbonate (0.3 eq, 0.39 mmol, 128 mg) and sodium iodide (6 mg). Themixture was heated to reflux for 7 days under Ar. The solvent wasevaporated under reduced pressure and the residue was taken inhexanes/ethyl acetate (ca 10%) and washed with water and brine. Theorganic layer was separated and dried over anhydrous sodium sulfate,filtered and evaporated under reduced to obtain an oil (1 g). Theresidue (1 g) was purified by gravity column chromatography on silicagel (MeOH in DCM, 0 to 4%). This gave compound III-14 as colorless oil(757 mg 0.99 mmol, 76%).

Example 89 Synthesis of Compound III-15

To a solution of 2-hexyldecyl 5-bromopentanoate (2 eq, 1.22 g, 3 mmol)in 15 ml of anhydrous THF (opened for 2 month), was added4-amino-1-butanol (1 eq, 1.5 mmol, 0.134 mg, 139 potassium carbonate (2eq, 3 mmol, 415 mg), cesium carbonate (0.3 eq, 0.45 mmol, 146 mg) andsodium iodide (6 mg). The mixture was heated to reflux for 6 days underargon.

Example 90 Synthesis of a Representative PEG Lipid

Pegylated lipid 90-6 (“PEG-DMA”) was prepared according to the abovereaction scheme, wherein n approximates the center of the range ofethylene oxide repeating units in the pegylated lipid.

Synthesis of 90-1 and 90-2

To a solution of myristic acid (6 g, 26 mmol) in toluene (50 mL) wasadded oxalyl chloride (39 mmol, 1.5 eq, 5 g) at room temperature. Afterthe resulting mixture was heated at 70° C. for 2 hours, the mixture wasconcentrated. The residue was taken up in toluene and concentratedagain. The residual oil was added via a syringe to a concentratedammonia solution (20 mL) at 10° C. The reaction mixture was filtered andwashed with water. The white solid was dried in vacuo. The desiredproduct was obtained as a white solid (3.47 g, 15 mmol, 58.7%).

Synthesis of 90-3

To suspension of 90-2 (3.47 g, 15 mmol) in THF (70 mL) was added inportions of lithium aluminum hydride (1.14 g, 30 mmol) at roomtemperature during 30 min period of time. Then the mixture was heated toreflux gently (oil bath at 65° C.) overnight. The mixture was cooled to5° C. and sodium sulfate 9 hydrate was added. The mixture was stirredfor 2 hours, filtered through a layer of celite, washed with 15% of MeOHin DCM (200 mL). The filtrate and washings were combined andconcentrated. The residual solid was dried in vacuo. The desired productwas obtained as a white solid (2.86 13.4 mmol, 89.5%).

Synthesis of 90-4

To a solution of myristic acid (3.86 g, 16.9 mmol) in benzene (40 mL)and DMF (1 drop) was added oxalyl chloride (25.35 mmol, 1.5 eq. 3.22 g)at room temperature. The mixture was stirred at room temperature for 1.5hours. Heated at 60° C. for 30 min. The mixture was concentrated. Theresidue was taken up in toluene and concentrated again. The residual oil(light yellow) was taken in 20 mL of benzene and added via syringe to asolution of 90-3 (2.86 13.4 mmol) and triethylamine (3.53 mL, 1.5 eq) inbenzene (40 mL) at 10° C. After addition, the resulting mixture wasstirred at room temperature overnight. The reaction mixture was dilutedwith water and was adjusted to pH 6-7 with 20% H₂SO₄. The mixture wasfiltered and washed with water. A pale solid was obtained. The crudeproduct was recrystallized from methanol. This gave the desired productas an off-white solid (5.65 g, 13 mmol, 100%).

Synthesis of 90-5

To suspension of 90-4 (5.65 g, 13 mmol) in THF (60 mL) was added inportions lithium aluminum hydride (0.99 g, 26 mmol) at room temperatureduring 30 min period of time. Then the mixture was heated to refluxgently overnight. The mixture was cooled to 0° C. and sodium sulfate 9hydrate. The mixture was stirred for 2 hours, then filtered through apad of celite and silica gel and washed with ether first. The filtrateturned cloudy and precipitation formed. Filtration gave a white solid.The solid was recrystallized from MeOH and a colorless crystalline solid(2.43 g).

The pad of celite and silica gel was then washed 5% of MeOH in DCM (400mL) and then 10% of MeOH in DCM with 1% of triethylamine (300 mL). Thefractions containing the desired product were combined and concentrated.A white solid was obtained. The solid was re-crystalized from MeOH and acolorless crystalline solid (0.79 g). The above two solids (2.43 g and0.79 g) were combined and dried in vacuo (3.20 g, 60%). ¹H NMR (CDCl₃ at7.27 ppm) 6: 2.58 (t-like, 7.2 Hz, 4H), 1.52-1.44 (m, 4H), 1.33-1.24 (m,44H), 0.89 (t-like, 6.6 Hz, 6H), 2.1-1.3 (very broad, 1H).

Synthesis of 90-6

To a solution of 92-5 (7 mmol, 2.87 g) and triethylamine (30 mmol, 4.18mL) in DCM (100 mL) was added a solution of mPEG-NHS (from NOF, 5.0mmol, 9.97 g, PEG MW approx. 2,000, n=about 45) in DCM (120 mL,). After24 h the reaction solution was washed with water (300 mL). The aqueousphase was extracted twice with DCM (100 mL×2). DCM extracts werecombined, washed with brine (100 mL). The organic phase was dried oversodium sulfate, filtered, concentrated partially. The concentratedsolution (ca 300 mL) was cooled at ca −15° C. Filtration gave a whitesolid (1.030 g, the unreacted starting amine). To the filtration wasadded Et₃N (1.6 mmol, 0.222 mL, 4 eq) and acetic anhydride (1.6 mmol,164 mg). The mixture was stirred at room temperature for 3 hours andthen concentrated to a solid. The residual solid was purified by columnchromatography on silica gel (0-8% methanol in DCM). This gave thedesired product as a white solid (9.211 g). ¹H NMR (CDCl₃ at 7.27 ppm)δ: 4.19 (s, 2H), 3.83-3.45 (m, 180200H), 3.38 (s, 3H), 3.28 (t-like, 7.6Hz, 2H, CH₂N), 3.18 (t-like, 7.8 Hz, 2H, CH2N), 1.89 (s, 6.6 H, water),1.58-1.48 (m, 4H), 1.36-1.21 (m, 48-50H), 0.88 (t-like, 6.6 Hz, 6H).

Example 91 Determination of pK_(a) of Formulated Lipids

As described elsewhere, the pKa of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). A representative preferred range of pKa is ˜5 to ˜7. The pK_(a)of each cationic lipid was determined in lipid nanoparticles using anassay based on fluorescence of 2-(p-toluidino)-6-napthalene sulfonicacid (TNS). Lipid nanoparticles comprising of cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) or a cationiclipid alone in PBS at a concentration of 0.4 mM total lipid are preparedusing the in-line process as described in Example 1. TNS was prepared asa 100 μM stock solution in distilled water. Vesicles were diluted to 24μM lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mMMES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranged from 2.5to 11. An aliquot of the TNS solution was added to give a finalconcentration of 1 μM and following vortex mixing fluorescence intensitywas measured at room temperature in a SLM Aminco Series 2 LuminescenceSpectrophotometer using excitation and emission wavelengths of 321 nmand 445 nm. A sigmoidal best fit analysis was applied to thefluorescence data and the pK_(a) was measured as the pH giving rise tohalf-maximal fluorescence intensity.

Example 92 Preparation of LNPs Comprising First and Second CationicLipids

LNPs comprising two different cationic lipids were prepared using thegeneral procedures set forth in Example 1. The LNPs included 0, 5, 10 or15 mole percent of a first cationic lipid (i.e., Cationic Lipid 1),while the second cationic lipid (i.e., Cationic Lipid 2) was maintainedat 10 mol percent in each LNP. Table 10 summarizes the ratio ofcomponents in each LNP and their properties.

TABLE 10 Properties of LNPs Cat lipid 1/Cat lipid 2/DSPC/ size MeanActivity Chol/PEGA* pKa (nm) PDI Encaps at 1 mg/kg SD 50/0/10/38.5/1.56.57 76 0.043 97 1628 680 45/5/10/38.5/1.5 6.42 73 0.053 97 2177 72840/10/10/38.5/1.5 6.29 70 0.004 97 5645 2498 35/15/10/38.5/1.5 6.16 710.002 98 3198 327

The data in Table 10 show that LNPs comprising only lipid 1, which has apKa outside the desirable range, have low activity, but activity of theLNPs can be surprisingly increased by combining cationic lipids 1 and 2.

Example 93 Synthesis of Compound IV-1

10-{[4′-(dimethylamino)butanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl2-hexyldecanoate

A solution of 10-[4′-(dimethylamino)butanoyl]oxynonadecan-1,19-diol(0.30 g), 2-hexyldecanoic acid (1.1 g),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.42 g) and4-dimethylaminopyridine (0.27 g) in dichloromethane (20 mL) was stirredat room temperature overnight. The solution was washed with dilutedhydrochloric acid, dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel (20 g)column with 0-8% methanol/dichloromethane. The desired product wasafforded (0.42 g).

Example 94 Synthesis of Compound IV-2

13-oxo-pentacosane-1,25-dioic acid

Sodium ethoxide (1.56 g) was dissolved in absolute ethanol (30 mL).Diethylacetone dicarboxylate (4.5 g) was added and the solution heatedto reflux. Ethyl 11-bromododecanoate (6.8 g) was slowly added and thesolution refluxed for an hour. Sodium ethoxide (1.53 g) was added,followed by ethyl 11-bromododecanoate (18 g). The solution was refluxedovernight. The reaction mixture was cooled, diluted with water,acidified with dilute hydrochloric acid, and extracted with methylenechloride. The organic fraction was washed with water and the solventremoved. The crude product was passed down a silica gel column (80 g)using methanol/methylene chloride to recover unreacted startingmaterials. The residue containing the product was treated with aceticacid (10 mL) and concentrated hydrochloric acid (20 mL), and thenrefluxed overnight. The solution was cooled, diluted with water andfiltered. The collected precipitate was recrystallized from acetone,affording the desired product as a white powder (2.9 g).

1,25-di-(2′-hexyldecyl) 13-oxo-pentacosanedioate

A solution of 13-oxo-pentacosane-1,25-dioic acid (0.91 g),4-dimethylaminopyridine (1.1 g),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.0 g) and2-hexyldecan-1-ol (2.4 g) in dichloromethane (40 mL) was stirred at roomtemperature overnight. The solution was washed with diluted hydrochloricacid, dried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel (20 g) column using0-4% ethyl acetate/hexane, affording the desired product (1 g).

1,25-di-(2′-hexyldecyl) 13-hydroxy-pentacosanedioate

A solution of 1,25-di-(2′-hexyldecyl) 13-oxo-pentacosanedioate (1 g) intetrahydrofuran (10 mL) and methanol (10 mL) was treated with sodiumborohydride (0.18 g). The reaction was stirred for 30 minutes and thendiluted with water, acidified and extracted with dichloromethane. Theorganic fraction was dried over anhydrous magnesium sulfate, filtered,and the solvent removed to afford the crude product (0.95 g). The crudeproduct was used in the next synthetic step without furtherpurification.

1,25-di-(2′-hexyldecyl)13-{[4-(dimethylamino)butanoyl]oxy}pentacosanedioate(Compound IV-2)

A solution of crude 1,25-di-(2′-hexyldecyl) 13-hydroxy-pentacosanedioate(0.95 g), 4-dimethylaminopyridine (0.42 g),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.34 g) andN,N-dimethylaminobutyric acid hydrochloride (0.59 g) in dichloromethane(15 mL) was stirred at room temperature for one hour. The solution waswashed with diluted hydrochloric acid followed by aqueous sodiumbicarbonate. The organic phase was dried over anhydrous magnesiumsulfate, filtered, and the solvent removed. The residue was passed downa silica gel (20 g) column using 0-6% methanol/dichloromethane to affordthe desired product (0.81 g).

Example 95 Synthesis of Compound IV-3

9-Tetrahydropyranyloxynonan-1-yl bromide

A solution of 9-bromononan-1-ol (25.6 g) and dihydropyran (10.5 g) indichloromethane (100 mL) was treated with pyridine p-toluenesulfonate(2.8 g) overnight. The solution was diluted with water and extractedwith dichloromethane. The organic fractions were dried over anhydrousmagnesium sulfate, filtered, and the solvent removed to afford thedesired crude product (35 g). The crude product was used in the nextsynthetic step without further purification.

1,19-Di(tetrahydropyranyloxy)nonadecan-10-ol

A solution of 9-tetrahydropyranyloxynonan-1-yl bromide (35 g) in diethylether (150 mL) was treated with magnesium (3.0 g). A crystal of iodinewas added to initiate the reaction. The solution was refluxed for 4days, then cooled to room temperature. Ethyl formate (4 mL) was slowlyadded and the solution refluxed for 2 hours. The solution was allowed tocool, then washed with dilute aqueous sulfuric acid. The organic phasewas washed with water, dried over anhydrous magnesium sulfate, filtered,and the solvent removed to yield 25 g of crude product. The crudematerial was added to 5% sodium hydroxide in a 1:10 water/methanolsolution (150 mL) and heated at 45° C. for one hour. The solution wascooled, diluted with water and extracted with hexane. The organicfractions were dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (200 g) columnusing 0-4% methanol/dichloromethane to afford the desired product (15g).

1,19-Di(tetrahydropyranyloxy)nonadecan-10-one

A solution of 1,19-di(tetrahydropyranyloxy)nonadecan-10-ol (7.2 g) indichloromethane (40 mL) was treated with pyridinium chlorochromate (4 g)and stirred overnight. Diethyl ether (200 mL) was added and the solutionfiltered through a silica gel bed. The solvent was removed and theresidue dissolved in hexane, then filtered through a silica gel bed. Thesolvent was removed and the residue passed down a silica gel (75 g)column using 0-3% methanol/dichloromethane to afford the desired product(3 g).

1,19-Dihydroxynonadecan-10-one

A solution of 1,19-di(tetrahydropyranyloxy)nonadecan-10-one (3 g) inmethanol (100 mL)/water (10 mL) was treated with pyridiniump-toluenesulfonate (1 g) overnight. The solution was filtered to affordthe desired product (0.8 g). Dilution of the filtrate with water,followed by extraction using dichloromethane afforded additional desiredproduct (1.0 g).

1,19-Di(2′hexyldecanoyloxy)nonadecan-10-one

A solution of 1,19-dihydroxynonadecan-10-one (0.87 g), 2-hexyldecanoicacid (2.48 g), 4-dimethylaminopyridine (1.0 g) andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.68 g) indichloromethane (40 mL) was stirred for three days. The solution wasdiluted with dichloromethane and washed with dilute hydrochloric acid.The organic fraction was dried over anhydrous magnesium sulfate,filtered, and the solvent removed. The residue was passed down a silicagel (50 g) column using dichloromethane to afford the desired product(2.8 g).

1,19-Di(2′hexyldecanoyloxy)nonadecan-10-ol

A solution of 1,19-di(2′hexyldecanoyloxy)nonadecan-10-one (1.06 g) indichloromethane (10 mL) was treated with sodium borohydride (0.15 g).Methanol was added dropwise until the materials dissolved. The solutionwas stirred for 30 minutes and then partitioned between water anddichloromethane. The organic phase was dried over anhydrous magnesiumsulfate, filtered and the solvent removed. The residue was passed down asilica gel (20 g) column using 0-12% ethyl acetate/hexane gradient toafford the desired product (0.40 g).

10-{[5-(dimethylamino)pentanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl2-hexyldecanoate (Compound IV-3)

A solution of 1,19-di(2′hexyldecanoyloxy)nonan-10-ol (0.40 g),5-N,N-dimethylaminopentanoic acid (0.22 g), 4-dimethylaminopyridine(0.18 g) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(0.18 g) in dichloromethane (20 mL) was stirred overnight. The solutionwas diluted with dichloromethane and washed with water. The organicfraction was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (20 g) columnusing 0-3% methanol/dichloromethane to afford the desired product (0.24g).

Example 96 Activity of Lipid Formulations Compared at DifferentComponent Ratios

Phosphatidylglycerols carry a formal negative charge in theirhead-groups, i.e., they are permanently negatively charged across therelevant physiological pH range. The cationic lipid in this instance hasa pKa of 6.57 which is higher than the expected ideal range whichsuggests it will produce an LNP that is more highly charged at neutralpH than would otherwise be wanted. Addition of small amounts of anioniccharged lipid provides a competing negative charge that is thought tobring the overall surface charge closer to neutrality. Specific resultsfor each respective anionic lipid are shown in Tables 11 and 12 below:

TABLE 11 Physical properties for LNPs formulated with DOPG.Encapsulation Mean Activity Cat lipid/DOPG/DSPC/Chol/PEGA size (nm) PDI(%) 1 mg/kg SD 50/0/10/38.5/1.5 72 0.043 97 6822 338750/0.5/9.5/38.5/1.5 69 0.015 96 5267 751 50/1/9/38.5/1.5 70 0.033 965051 160 50/2.5/7.5/38.5/1.5 67 0.028 96 7228 826 50/5/5/38.5/1.5 640.051 95 8417 400 50/10/0/38.5/1.5 69 0.047 95 5979 387350/1/10/37.5/1.5 70 0.030 96 8256 1229 50/5/10/33.5/1.5 63 0.077 94 70351735 50/10/10/28.5/1.5 59 0.042 91 4081 2463

Table 11 shows that addition of a negatively chargedphosphatidylglycerol provides a benefit. It is also shown that theoptimum for the addition of phosphatidylglycerol lipid moves to a lowerrange if the DSPC structural lipid is held constant.

TABLE 12 Physical properties for LNPs formulated with DSPG. size EncapsMean Activity Cat lipid/DSPG/DSPC/Chol/PEGA (nm) PDI (%) 1 mg/kg SD50/0/10/38.5/1.5 67 0.045 98 3627 1800 50/1/9/38.5/1.5 67 0.021 97 62523597 50/2.5/7.5/38.5/1.5 67 0.096 95 7500 5560 50/5/5/38.5/1.5 58 0.02696 4033 2179 50/7.5/2.5/38.5/1.5 57 0.040 94 559 407 50/10/0/38.5/1.5 640.131 91 152 72 50/1/10/37.5/1.5 68 0.026 97 4594 1382 50/5/10/33.5/1.565 0.157 97 2243 1386 50/10/10/28.5/1.5 55 0.062 96 411 410

For comparison, a similar series described in Table 12 was generatedusing DSPG instead of DOPG, i.e., the same anionically charged headgroupbut lipid tails that match DSPC. The results show a similar trend whenDSPG is compared to DOPG. Specifically, there is a benefit to theaddition of DSPG and that maximum benefit is achieved at lower levels ifthe DSPG is added in place of the steroid rather than the neutral lipid(e.g., DSPC). These results demonstrate the utility ofphospatidylglycerols in general, the interplay of the other lipidcomponents, and demonstrate the surprising benefit and utility ofphosphatidylglycerols lipids.

Example 97 Variable mRNA to Lipid Ratios and Results

The effect of the proportion of mRNA to total lipid was investigated fora particular standard set of lipid proportions across threerepresentative cationic lipids. LNP's were formulated by an in-linemixing process as described in Example 1. The lipid proportions wereheld constant at 47.5:10:40.7:1.8 for the cationic lipid, DSPC,Cholesterol and PEG-lipid respectively. The mRNA to lipid ratios aredescribed in terms of the N/P ratio where N represents the moles ofcationic lipid and P represents the moles of phosphate present as partof the nucleic acid backbone. Ratios described in this way areindependent of the size of the nucleic acid. The size and polydispersityindex data were generated using Malvern Nanosizer ZS. The diametersgiven are intensity weighted means. Encapsulation was determined using afluorescent intercalating dye based assay (Ribogreen). Activity data wasgenerated using an in vivo murine model of mRNA expression based onphotinus pyralis (i.e., firefly) luciferase as described in Example 2using a dose of 1 mg/kg.

Table 13 summarizes results for each of three representative cationiclipids (compound I-5, II-9, III-45), and the luciferase activity data isplotted in FIGS. 1 and 2.

TABLE 13 Summary of Physical Characteristics and Luciferase Activity forrepresentative LNP formulations with varying N/P ratio Encapsu- LiverLuc Spleen Luc Cationic lation Size Activity Activity Lipid N/P (%) (nm)PDI ng/g ng/g I-5  3:1 94 86 0.072 7689 188  6:1 96 81 0.028 9509 23612:1 98 75 0.026 12826 184 22:1 99 81 0.039 14538 247 II-9  3:1 92 940.054 6727 831  6:1 95 88 0.033 12209 642 12:1 98 84 0.014 18330 51422:1 98 84 0.017 12634 439 III-45  3:1 75 87 0.089 13308 410  6:1 86 740.068 15395 356 12:1 91 65 0.074 16596 243 22:1 94 57 0.083 16005 353

1. A lipid nanoparticle comprising: i) from 47 to 48 mol percent of acationic lipid; ii) a neutral lipid; iii) a steroid; iv) a polymerconjugated lipid; and v) a therapeutic agent, or a pharmaceuticallyacceptable salt thereof, encapsulated within or associated with thelipid nanoparticle. 2-9. (canceled)
 10. The lipid nanoparticle of claim1, comprising from 47.2 to 47.8 mol percent of the cationic lipid. 11.(canceled)
 12. The lipid nanoparticle of claim 1, wherein the neutrallipid is present in a concentration ranging from 5 to 15 mol percent.13-15. (canceled)
 16. The lipid nanoparticle of claim 1, wherein thesteroid is present in a concentration ranging from 32 to 40 mol percent.17-18. (canceled)
 19. The lipid nanoparticle of claim 1, wherein themolar ratio of the cationic lipid to the steroid ranges from 1.0:0.9 to1.0:1.2.
 20. (canceled)
 21. A lipid nanoparticle comprising: i) acationic lipid having an effective pKa greater than 6.0; ii) from 5 to15 mol percent of a neutral lipid; iii) from 1 to 15 mol percent of ananionic lipid; iv) from 30 to 45 mol percent of a steroid; v) a polymerconjugated lipid; and vi) a therapeutic agent, or a pharmaceuticallyacceptable salt or prodrug thereof, encapsulated within or associatedwith the lipid nanoparticle, wherein the mol percent is determined basedon total mol of lipid present in the lipid nanoparticle. 22-30.(canceled)
 31. The lipid nanoparticle of claim 1, wherein the cationiclipid has a structure of Formula I:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,—S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other ofL¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond; R^(a) is H or C₁-C₁₂ alkyl; R^(1a) andR^(1b) are, at each occurrence, independently either (a) H or C₁-C₁₂alkyl, or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(1b) and the carbon atom to which it is bound to form a carbon-carbondouble bond; R^(2a) and R^(2b) are, at each occurrence, independentlyeither (a) H or C₁-C₁₂ alkyl, or (b) R^(2a) is H or C₁-C₁₂ alkyl, andR^(2b) together with the carbon atom to which it is bound is takentogether with an adjacent R^(2b) and the carbon atom to which it isbound to form a carbon-carbon double bond; R^(3a) and R^(3b) are, ateach occurrence, independently either (a) H or C₁-C₁₂ alkyl, or (b)R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(3b) and thecarbon atom to which it is bound to form a carbon-carbon double bond;R^(4a) and R^(4b) are, at each occurrence, independently either (a) H orC₁-C₁₂ alkyl, or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond; R⁵ and R⁶ are each independently methyl orcycloalkyl; R⁷ is, at each occurrence, independently H or C₁-C₁₂ alkyl;R⁸ and R⁹ are each independently unsubstituted C₁-C₁₂ alkyl; or R⁸ andR⁹, together with the nitrogen atom to which they are attached, form a5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a andd are each independently an integer from 0 to 24; b and c are eachindependently an integer from 1 to 24; e is 1 or 2; and x is 0, 1 or 2.32. The lipid nanoparticle of claim 1, wherein the cationic lipid has astructure of Formula II:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,—S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other ofL¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond; G¹ is C₁-C₂ alkylene,—(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)— or a direct bond; G² is—C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct bond; G³ is C₁-C₆alkylene; R^(a) is H or C₁-C₁₂ alkyl; R^(1a) and R^(1b) are, at eachoccurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a)is H or C₁-C₁₂ alkyl, and R^(1b) together with the carbon atom to whichit is bound is taken together with an adjacent R^(1b) and the carbonatom to which it is bound to form a carbon-carbon double bond; R^(2a)and R^(2b) are, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence,independently either (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H orC₁-C₁₂ alkyl, and R^(3b) together with the carbon atom to which it isbound is taken together with an adjacent R^(3b) and the carbon atom towhich it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b)are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or(b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbonatom to which it is bound is taken together with an adjacent R^(4b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond; R⁵ and R⁶ are each independently H or methyl; R⁷ is C₄-C₂₀ alkyl;R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹, togetherwith the nitrogen atom to which they are attached, form a 5, 6 or7-membered heterocyclic ring; a, b, c and d are each independently aninteger from 1 to 24; and x is 0, 1 or
 2. 33. The lipid nanoparticle ofclaim 1, wherein the cationic lipid has a structure of Formula III:

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,—S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other ofL¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond; G¹ and G² are eachindependently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ isC₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈cycloalkenylene; R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are eachindependently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R³ is H, OR^(S), CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H orC₁-C₆ alkyl; and x is 0, 1 or
 2. 34. The lipid nanoparticle of claim 1,wherein the cationic lipid has the following Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond; L is, at eachoccurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X; X isCR^(a); Z is alkyl, cycloalkyl or a monovalent moiety comprising atleast one polar functional group when n is 1; or Z is alkylene,cycloalkylene or a polyvalent moiety comprising at least one polarfunctional group when n is greater than 1; R^(a) is, at each occurrence,independently H, C₁-C₁₂ alkyl, C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl,C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl,C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂alkylcarbonyl; R is, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond; R¹ and R² have, at eachoccurrence, the following structure, respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c²are, at each occurrence, independently an integer from 5 to 10; d¹ andd² are, at each occurrence, independently an integer from 5 to 10; y is,at each occurrence, independently an integer from 0 to 2; and n is aninteger from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl,aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl,alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionallysubstituted with one or more substituent.
 35. The lipid nanoparticle ofclaim 1, wherein the cationic lipid has the following Formula (V):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond; L is, at eachoccurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X; X isCR^(a); Z is alkyl, cycloalkyl or a monovalent moiety comprising atleast one polar functional group when n is 1; or Z is alkylene,cycloalkylene or a polyvalent moiety comprising at least one polarfunctional group when n is greater than 1; R^(a) is, at each occurrence,independently H, C₁-C₁₂ alkyl, C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl,C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl,C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂alkylcarbonyl; R is, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond; R¹ and R² have, at eachoccurrence, the following structure, respectively:

R′ is, at each occurrence, independently H or C₁-C₁₂ alkyl; a¹ and a²are, at each occurrence, independently an integer from 3 to 12; b¹ andb² are, at each occurrence, independently 0 or 1; c¹ and c² are, at eachoccurrence, independently an integer from 2 to 12; d¹ and d² are, ateach occurrence, independently an integer from 2 to 12; y is, at eachoccurrence, independently an integer from 0 to 2; and n is an integerfrom 1 to 6, wherein a¹, a², c¹, c², d¹ and d² are selected such thatthe sum of a¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d²is an integer from 18 to 30, and wherein each alkyl, alkylene,hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl andalkylcarbonyl is optionally substituted with one or more sub stituent.36. (canceled)
 37. The lipid nanoparticle of claim 1, wherein thecationic lipid has one of the following structures:


38. The lipid nanoparticle of claim 1, wherein the cationic lipid hasthe following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkenyl, optionally substituted C₁₀-C₃₀ alkynyl or optionallysubstituted C₁₀-C₃₀ acyl; R₃ is H, optionally substituted C₁₀-C₁₀ alkyl,optionally substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,alkylphosphorodithioate, alkylphosphonate, alkylamine, hydroxyalkyl,ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,ω-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, mw100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl, orheterocycle, or linker-ligand; and E is O, S, N(Q), C(O), N(Q)C(O),C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)₂N(Q), S(O)₂, N(Q)S(O)₂, SS,O═N, aryl, heteroaryl, cyclic or heterocycle; and Q is H, alkyl,ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl orω-thiophosphoalkyl.
 39. The lipid nanoparticle of claim 1, wherein thecationic lipid has one of the following structures:


40. The lipid nanoparticle of claim 1, wherein the molar ratio ofcationic lipid to the neutral lipid ranges from about 4.1:1.0 to about4.9:1.0. 41-43. (canceled)
 44. The lipid nanoparticle of claim 1,wherein the molar ratio of cationic lipid to steroid ranges from 5:1 to1:1.
 45. (canceled)
 46. The lipid nanoparticle of claim 1, wherein themolar ratio of cationic lipid to polymer conjugated lipid ranges fromabout 100:1 to about 20:1.
 47. (canceled)
 48. A lipid nanoparticlecomprising: i) a first cationic lipid having a first effective pKa; ii)a second cationic lipid having a second effective pKa, the secondeffective pKa being greater than the first effective pKa; iii) a neutrallipid; iv) a steroid; v) a polymer conjugated lipid; and vi) atherapeutic agent, or a pharmaceutically acceptable salt or prodrugthereof, encapsulated within or associated with the lipid nanoparticle,wherein the lipid nanoparticle has an effective pKa between the firstand second effective pKa's. 49-68. (canceled)
 69. The lipid nanoparticleof claim 1, wherein the neutral lipid is di stearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE) or 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). 70-71. (canceled)
 72. The lipid nanoparticle of claim 1,wherein the steroid is cholesterol.
 73. The lipid nanoparticle of claim1, wherein the polymer conjugated lipid is present in a concentrationranging from 1.0 to 2.5 molar percent. 74-75. (canceled)
 76. The lipidnanoparticle of claim 1, wherein the polymer conjugated lipid is apegylated lipid.
 77. The lipid nanoparticle of claim 76, wherein thepegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEGdialkyoxypropylcarbamate.
 78. The lipid nanoparticle of claim 76,wherein the pegylated lipid has the following Formula (VI):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R¹² and R¹³ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and w has a mean value ranging from 30 to
 60. 79-81.(canceled)
 82. The lipid nanoparticle of claim 78, wherein the pegylatedlipid has the following Formula (VIa):

wherein the average w is about
 49. 83. The lipid nanoparticle of claim1, wherein the therapeutic agent comprises a nucleic acid.
 84. The lipidnanoparticle of claim 83, wherein the nucleic acid is selected fromantisense and messenger RNA.
 85. The lipid nanoparticle of claim 1,wherein a plurality of the nanoparticles has a polydispersity of lessthan 0.12. 86-88. (canceled)
 89. A pharmaceutical composition comprisinga lipid nanoparticle of claim 1 and a pharmaceutically acceptableexcipient.
 90. A method for administering a therapeutic agent to apatient in need thereof, the method comprising administering the lipidnanoparticle of claim 1 to the patient.
 91. A method for treating adisease in a patient in need thereof, the method comprisingadministering the lipid nanoparticle of claim 1 to the patient, whereinthe therapeutic agent is effective to treat the disease.